Methods of administering 3,4-diaminopyridine

ABSTRACT

Provided herein are methods of determining NAT acetylation status of a subject with a 3,4-DAP-sensitive disease, methods of selecting a dose of 3,4-DAP or a pharmaceutically acceptable salt thereof adjusted to a subject&#39;s acetylation status, methods of administering 3,4-diaminopyridine or a pharmaceutically acceptable salt thereof to a patient in need thereof, and methods of treating 3,4-DAP sensitive diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.61/503,553, filed Jun. 30, 2011; and 61/553,045, filed Oct. 28, 2011;the disclosure of each of which is incorporated herein by reference inits entirety.

FIELD

Provided herein are methods of determining NAT acetylation status of asubject with a 3,4-DAP-sensitive disease, methods of selecting a dose of3,4-DAP or a pharmaceutically acceptable salt thereof adjusted to asubject's acetylation status, methods of administering3,4-diaminopyridine or a pharmaceutically acceptable salt thereof to apatient in need thereof, and methods of treating 3,4-DAP sensitivediseases.

BACKGROUND

It has been described that 3,4-diaminopyridine can be used for thetreatment of myasthenia gravis and myasthenic syndromes (includingLambert-Eaton myasthenic syndrome, congenital myasthenia, and myasthenicsyndromes of medicinal or toxic origin) because it improvesneuromuscular transmission by increasing the entry of cellular calcium,which promotes the release of acetylcholine in the nerve endings (MurrayN. M. et al., Neurology 1981, 31, 265-27; McEvoy K. M. et al., N. Engl.J. Med. 1989, 321, 1567-1571). The ability of 3,4-diaminopyridine toincrease the release of acetylcholine in the nerve endings also makes itpossible to envisage its use in improving the cognitive functions duringaging (U.S. Pat. No. 4,386,095). 3,4-Diaminopyridine may also be usefulfor the symptomatic treatment of fatigue related to a neurologicalpathology, such as, for example, multiple sclerosis (Bever et al.,Annals of Neurology 1990, 27, 421-427 and Sheean et al., Brain 1998,121, 967-975). Finally the use of 3,4-diaminopyridine has been describedfor the treatment of diseases affecting motor neuron cells, such asacute infectious poliomyelitis and its effects, Creutzfeldt-Jakobsyndrome, some toxic and nutritional disorders, such as those related tovitamin B deficiency, degeneration of motor neurons as a result ofexposure to certain compounds, such as aluminum, or degenerativediseases, such as amyotrophic lateral sclerosis, primary lateralsclerosis, pre-senile dementia with attack on motor neurons, spinalmuscular atrophies, olivoponto-cerebellar atrophy, Joseph's disease,Parkinson's disease, Huntington's chorea or Pick's disease.

Lambert-Eaton myasthenic syndrome (LEMS) is a rare autoimmune diseasewith the primary symptom of proximal muscle weakness. Muscle weaknessresulting from LEMS is caused by auto-antibodies to voltage-gatedcalcium channels leading to a reduction in the amount of acetylcholinereleased from nerve terminals. The first clinical symptom of LEMS istypically proximal muscle weakness that may impact ability to walk andclimb stairs.

The treatment options available for patients with LEMS can be classifiedinto 3 main categories, each targeting different aspects of thepathogenesis of the disease: 1) anti-tumor treatment (e.g.,chemotherapy) in the paraneoplastic form, 2) immunologic treatments(e.g., intravenous immunoglobulin (IVIG), plasma exchange,immunoadsorption, prednisone, azathioprine) suppressing the autoimmunereaction, and 3) symptomatic treatment (e.g., pyridostigmine,amifampridine). The treatment strategies may include the above optionsindividually or in different combinations; however, there are no datafrom published randomized controlled trials to support the use of thevarious combinations.

The applicants have demonstrated that the pharmacokinetic profile oforally administered 3,4-DAP is highly variable between patients with upto 10-fold differences in maximum observed plasma concentration(C_(max)), area under the plasma concentration-time curve (AUC) andapparent plasma terminal elimination half-life (t_(1/2)) betweensubjects. Similar variability has been demonstrated in the literature(Wirtz et al., Nature 2009, 86(1), 44-48; Bever et al., Ann Neurol 1990,27, 421-427; and Bever et al., Neurology 1996, 47, 1457-1462). However,no rationale has been provided previously for this variability. Further,no methods have been described in the literature for determining how apatient will respond to 3,4-DAP without actually administering 3,4-DAPwith the attendant risks of side effects and lack of efficacy. Thisvariability suggests that the method of administering and/or amount of3,4-DAP should be adjusted to each patient. Thus, there exists a needfor alternative and improved methods of administering3,4-diaminopyridine.

SUMMARY

Provided herein are methods of determining NAT acetylation status of asubject with a 3,4-DAP-sensitive disease, methods of selecting a dose of3,4-DAP or a pharmaceutically acceptable salt thereof adjusted to asubject's acetylation status, methods of administering3,4-diaminopyridine or a pharmaceutically acceptable salt thereof to apatient in need thereof, and methods of treating 3,4-DAP sensitivediseases, in a manner that improves or maximizes its efficacy, and/orimproves or maximizes its oral bioavailability, and/or improves oroptimizes the consistency of oral bioavailability from oneadministration to the next, and/or decreases the frequency or severityof adverse events. Such methods can be applied in the treatment of any3,4-diaminopyridine-responsive disorder, including, but not limited to,myasthenia gravis and myasthenic syndromes (including Lambert-Eatonmyasthenic syndrome, congenital myasthenia, and myasthenic syndromes ofmedicinal or toxic origin), multiple sclerosis, improving the cognitivefunctions during aging, treatment of fatigue related to a neurologicalpathology, diseases affecting motor neuron cells, such as acuteinfectious poliomyelitis and its effects, Creutzfeldt-Jakob syndrome,some toxic and nutritional disorders, such as those related to vitamin Bdeficiency, degeneration of motor neurons as a result of exposure tocertain compounds, such as aluminum, or degenerative diseases, such asamyotrophic lateral sclerosis, primary lateral sclerosis, pre-seniledementia with attack on motor neurons, spinal muscular atrophies,olivoponto-cerebellar atrophy, Joseph's disease, Parkinson's disease,Huntington's chorea or Pick's disease. The methods provided hereinadvantageously allow better control of clinical symptoms, e.g., fewerand/or less severe adverse events or other clinical parameters.

As used herein, 3,4-DAP refers to 3,4-diaminopyridine and amifampridine.The term 3,4-DAP as used herein includes a pharmaceutically acceptablesalt of 3,4-diaminopyridine, unless the context dictates otherwise.3-N-AcDAP, 3-N-acetyl DAP, and 3-N-acetyl amifampridine refer toN-(4-aminopyridin-3-yl)acetamide.

In a first aspect, provided herein is a method comprising administering3,4-diaminopyridine or a pharmaceutically acceptable salt thereof, to ahuman in need thereof, and informing said human that the frequencyand/or severity of side effect(s) of said 3,4-diaminopyridine orpharmaceutically acceptable salt thereof are decreased when it isingested with food compared to when ingested without food.

In a second aspect, provided herein is a method comprising determiningwhether a subject is a slow acetylator or fast acetylator of 3,4-DAP.

In a third aspect, provided herein is a method of selecting a dose of3,4-DAP or a pharmaceutically acceptable salt thereof in an amount thatis adjusted to the subject's acetylator status.

In a fourth aspect, provided herein is a method of administering a doseof 3,4-DAP or a pharmaceutically acceptable salt thereof in an amountthat is adjusted to the subject's acetylator status.

In a fifth aspect, provided herein is a method of selecting a dose of3,4-DAP or a pharmaceutically acceptable salt thereof for a subject whois a fast acetylator and informing the fast acetylator to take said3,4-DAP without food.

In a sixth aspect, provided herein is a method of treating a3,4-DAP-sensitive disease comprising determining whether a subject is aslow acetylator or fast acetylator; selecting a dose of 3,4-DAP or apharmaceutically acceptable salt thereof based on the subject'sacetylator status; and administering the dose of 3,4-DAP to the subjectin need thereof.

In a seventh aspect, provided herein is a method of treating a3,4-DAP-sensitive disease comprising administering a dose of 3,4-DAP tothe subject in need thereof in an amount adjusted to the subject'sacetylator status.

In an eighth aspect, provided herein is a method of treating a3,4-DAP-responsive disorder with 3,4-DAP or a pharmaceuticallyacceptable salt thereof in a subject who is a fast acetylator in a doseof 80 mg or more per day.

In a ninth aspect, provided herein is a method of treating a3,4-DAP-responsive disorder with 3,4-DAP or a pharmaceuticallyacceptable salt thereof in combination with an inhibitor of an NATenzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows concentrations of 3,4-diaminopyridine and its metaboliteN-(4-aminopyridin-3-yl)acetamide in rat hepatocytes for the experimentdescribed in Example 1.

FIG. 2 shows concentrations of 3,4-diaminopyridine in dog hepatocytesfor the experiment described in Example 2.

FIG. 3 shows concentrations of 3,4-diaminopyridine and its metaboliteN-(4-aminopyridin-3-yl)acetamide in human hepatocytes for the experimentdescribed in Example 3.

FIG. 4 shows the concentrations of 3,4-diaminopyridine and itsmetabolite N-(4-aminopyridin-3-yl)acetamide produced in vivo in ratsafter the first dose in a fasted state at various doses (2, 8, or 25mg/kg/dose) for the experiment described in Example 4.

FIG. 5 shows the C_(max) and AUC ratio of the metaboliteN-(4-aminopyridin-3-yl)acetamide to 3,4-diaminopyridine in vivo in ratsafter the first dose in a fasted state for the experiment described inExample 4.

FIG. 6 shows the mean plasma concentrations (+SD) of 3,4-DAP andN-(4-aminopyridin-3-yl)acetamide following oral administration ofamifampridine phosphate to all subjects in a fed state for the clinicaltrial described in Example 10.

FIG. 7 shows the mean plasma concentrations (+SD) of 3,4-DAP andN-(4-aminopyridin-3-yl)acetamide following oral administration of3,4-DAP phosphate to all subjects in a fasted state for the clinicaltrial described in Example 10.

FIG. 8 shows the human plasma 3,4-diaminopyridine plasma concentrationat four hours post dose in a fasted state for the clinical trialdescribed in Example 10.

FIG. 9 shows the 3,4-diaminopyridine phosphate exposure in two humanpatients after a single oral dose in a fasted state for the clinicaltrial described in Example 10.

FIG. 10 depicts mean plasma concentrations (+SD) of 3,4-DAP followingoral administration of 3,4-DAP phosphate to all subjects in a fed orfasted state for the clinical trial described in Example 10.

FIG. 11 depicts mean PK parameters for 3,4-DAP in fed and fastedsubjects following administration of oral 3,4-DAP phosphate for theclinical trial described in Example 10.

FIG. 12 depicts mean plasma concentrations (+SD) ofN-(4-aminopyridin-3-yl)acetamide following oral administration of3,4-DAP phosphate to all subjects in a fed or fasted state for theclinical trial described in Example 10.

FIG. 13 depicts mean pharmacokinetic parameters forN-(4-aminopyridin-3-yl)acetamide in fed and fasted subjects followingadministration of oral 3,4-DAP phosphate for the clinical trialdescribed in Example 10.

FIG. 14 depicts mean, maximal and minimal urinary excretion of 3,4-DAPand N-(4-aminopyridin-3-yl)acetamide in fed and fasted subjectsreceiving oral 3,4-DAP phosphate for the clinical trial described inExample 10.

FIG. 15 shows the schedule of events for the evaluation of safety forthe clinical trial described in Example 10.

FIG. 16 depicts mean PK parameters (arithmetic values) for treatmentgroups A and B in the clinical trial described in Example A.

FIG. 17 depicts mean values of 3,4-DAP phosphate or free base (ng/mL) attime sampling for the clinical trial described in Example A.

FIGS. 18a and 18b depict 3,4-DAP as phosphate salt or free base:arithmetic means for the clinical trial described in Example A.

FIG. 19a depicts 90% CI for AUCs and C_(max) free base/phosphate saltratio for the clinical trial described in Example A.

FIG. 19b depicts time to reach C_(max) for the clinical trial describedin Example A.

FIG. 20 depicts concentration response data for 3,4-DAP Phosphate andN-(4-aminopyridin-3-yl)acetamide HCl in CHO Cells TransientlyTransfected with hKv1.7 for the experiment described in Example 5.

FIG. 21a and FIG. 21b depict concentration response data for 3,4-DAPphosphate and N-(4-aminopyridin-3-yl)acetamide HCl in CHO or HEK293Cells transiently transfected with hKv1.1, hKv1.2, hKv1.3, hKv1.4, orhKv1.5 for the experiment described in Example 6.

FIG. 22 depicts 3,4-DAP C_(max) for slow and fast acetylators for theTrial Described in Example 18, Part 1.

FIG. 23 depicts 3,4-DAP AUC_(0-t) for slow and fast acetylators for thetrial described in Example 18, Part 1.

FIGS. 24a and 24b depict mean pharmacokinetic parameters (±SD) of3,4-DAP and 3-N-acetyl (5-30 mg 3,4-DAP base equivalents) in healthysubjects after single oral doses of Firdapse® in slow and fastacetylator phentoypes for the trial described in Example 18, Part 1.

FIGS. 25a, 25b, 25c, and 25d depict mean plasma concentration-timeprofiles of BMN125 after single oral doses of Firdapse® at 5, 10, 20 and30 mg (base equivalents), respectively, in healthy subjects with slowand fast acetylator phenotypes for the trial described in Example 18,Part 1.

FIGS. 26a, 26b, and 26c depict mean plasma concentrations of 3,4-DAP inslow and fast acetylators, QID Dosing of Firdapse® (20 mg base equiv),Day 1, 3, and 4, respectively, for the trial described in Example 18,Part 2.

FIGS. 27a, 27b, and 27c depict mean plasma concentrations ofN-(4-aminopyridin-3-yl)acetamide in slow and fast acetylators, QIDdosing of Firdapse® (20 mg base equiv) on Day 1, 3, and 4, respectively,for the trial described in Example 18, Part 2.

FIG. 28 depicts treatment-emergent drug-related adverse events bytreatment and phenotype for Part 1 of the Study in Example 18.

FIG. 29 depicts treatment-emergent drug-related adverse events bytreatment and phenotype for Part 2 of the study in Example 18.

DETAILED DESCRIPTION

Provided herein are methods of administering a purified preparation of3,4-diaminopyridine (3,4-DAP, amifampridine) including apharmaceutically acceptable salt thereof. The Applicants have found thatthe PK profile of administered 3,4-DAP is highly variable betweenpatients with up to 10-fold differences in maximum observed plasmaconcentration (C_(max)), area under the plasma concentration-time curve(AUC) and apparent plasma terminal elimination half-life (t_(1/2))between subjects (Example A, FIG. 16). The Applicants have discoveredthat the variability between patients is explained by the surprisingmetabolic disposition of amifampridine through N-acetyl transferases(NAT) to form the major 3-N-acetyl metabolite, depicted below

3-N-acetyl-3,4-diaminopyridine, and herein identified for the firsttime.

NAT enzymes are present in most mammalian species. However, dogs lackNAT and acetylation pathways entirely. In mouse and rat, NAT1, NAT2,NAT3 subtypes are present. In humans, NAT1 and NAT2 subtypes arepresent. NAT2 is located primarily in liver and intestine and NAT1 isubiquitous and present in virtually all tissues. NAT enzymes are highlypolymorphic in humans, and individuals can be classified as slow andrapid acetylators (Casarett & Doull's Toxicology, The Basic Science ofPoisons 7th Ed. (2008) Chapter 6: Biotransformation of Xenobiotics. pp.278-282). The polymorphism of the NAT enzymes is well characterized inthe Caucasian population, where the slow acetylator phenotype is presentin 50-59% of Caucasians (Casarett & Doull's Toxicology, The BasicScience of Poisons 7th Ed. (2008) Chapter 6: Biotransformation ofXenobiotics. pp. 278-282). Acetylator subtype varies considerably withethnicity (Casarett & Doull's Toxicology, The Basic Science of Poisons7th Ed. (2008) Chapter 6: Biotransformation of Xenobiotics. pp.278-282). An individual's acetylator phenotype can be determined byusing procedures described in Example 15 and Example 15a. Anindividual's acetylator genotype can be determined by using proceduresdescribed in Example 16. For isoniazid, a half life of 1 to 2 hoursindicates a rapid acetylator and a half life of 2 to 5 hours indicates aslow acetylator. Acetylator phenotype affects the overall PK parametersand disposition of amifampridine in individual humans. In particular,high C_(max) in animals (including, but not limited to fasted rats) hascorrelated to toxicity.

In vitro comparative metabolism studies conducted in hepatocytesdemonstrated the formation of a single major acetylated amifampridinemetabolite in rat (extensive) and human hepatocytes (variable) (Examples1 and 3, respectively and FIGS. 1 and 3, respectively), but not dog(Example 2 and FIG. 2). This is consistent with the lack of the N-acetyltransferase (NAT) enzyme and acetylation pathway in dog. Thus, the dogwith its lack of amifampridine metabolism (corresponding to the slowhuman acetylator population) and the rat with its rapid rate ofmetabolism (corresponding to the fast human acetylator population) arerepresentative models for parent and metabolite exposure in humans. Asindicated in FIGS. 1 and 3, ¹⁴C-3,4-diaminopyridine phosphate rat andhuman hepatocytes metabolism studies indicate conversion toN-(4-aminopyridin-3-yl)acetamide. No conversion of 3,4-DAP to itsmetabolite was seen in dog hepatocytes (FIG. 2). In multiple humanhepatocyte donor incubations variable metabolism/conversionN-(4-aminopyridin-3-yl)acetamide is observed (FIG. 3, for example).

In a rat pharmacokinetic study (Example 4, FIGS. 4 and 5), it wasdemonstrated that the N-(4-aminopyridin-3-yl)acetamide metabolite, themajor circulating metabolite, is more abundant than 3,4-DAP and is amodel for rapid metabolism. Exposure to N-(4-aminopyridin-3-yl)acetamidedemonstrates a dose related dependence and a first pass effect (liver).In vitro metabolic studies in rat hepatocytes (Example 1) correctlypredict in vivo observations. Additional in vitro pharmacodynamicstudies indicate the N-(4-aminopyridin-3-yl)acetamide metabolite isinactive in the evaluated K+ channels (FIGS. 20 and 21).

In a human clinical trial (Example 10), N-(4-aminopyridin-3-yl)acetamideconcentration in vivo is significantly higher than 3,4-DAP at all timepoints (FIG. 6, fed state and FIG. 7, fasted state) and with high levelof variability (FIG. 8). This is the first demonstration in human that3,4-diaminopyridine undergoes extensive metabolic conversion to themajor circulating metabolite N-(4-aminopyridin-3-yl)acetamide.

FIG. 6 depicts the concentration in vivo of 3,4-DAP and its metaboliteN-(4-aminopyridin-3-yl)acetamide in a fed state. FIG. 7 depicts theconcentration in vivo of 3,4-DAP and its metaboliteN-(4-aminopyridin-3-yl)acetamide in a fasted state. A comparison ofFIGS. 6 and 7 demonstrate that administering 3,4-DAP with food reducesthe concentration of the compound which reduces the potential forassociated side effects.

In the clinical study described in Example 10, the 3,4-DAP plasma levelsat 4 hours post dose suggests emerging bimodal distribution by plasmaconcentration in fasted subjects (FIG. 8). Subjects in this study werenot tested to determine whether they were fast or slow metabolizers.Retrospectively, fast acetylators were likely in the region of 0-5 ng/mL3,4-DAP at 4 hours post dose and slow acetylators were likely in thebroad region of 10-26 ng/mL 3,4-DAP at 4 hours post dose. Intermediateacetylators were likely in the region of 5-10 ng/mL at 4 hours postdose. Intermediate acetylators are categorized as fast acetylators.Examples of PK for two patients from Example 10 after a single oral doseof 3,4-DAP are depicted in FIG. 9. Subject A was likely to be a fastacetylator with a 3,4-DAP phosphate half life of 74 min and a lowC_(max) of 18 ng/mL for measurable 3,4-DAP phosphate levels of up to 4hours post dose. Subject B was likely to be a slow acetylator with a3,4-DAP phosphate half life of 152 min and a C_(max), of 138 ng/mL formeasurable 3,4-DAP phosphate levels of up to 12 hours post dose. High PKvariability poses significant issues for attempting to achieve maximumefficacy and safety. Dosing subject B too often (e.g., more than once aday) and/or with too much drug has the potential for increased sideeffects. Dosing subject A too infrequently and/or with too little drug(e.g., only once or twice a day) has the potential for reduced or lackof efficacy thus delaying relief for a patient from the symptoms oftheir disease.

In a second study described in Example 18, it was confirmed that thereare slow and fast acetylators of 3,4-DAP. See Table 5 for phenotypingand genotyping data for each subject in Part 1 and 2 of the trial inExample 18 which data categorizes each subject as slow or fast. In Part1 of the study in Example 18, across each of the single dose groups, theratio of mean C_(max) values for slow acetylators ranged from 3.5 to 4.5fold greater than for fast acetylators (FIG. 22 and FIG. 24)demonstrating that there are large differences in metabolism amongsubjects.

Slow acetylators have higher plasma levels, exaggerated pharmacology,and more severe and/or more frequent adverse events (FIG. 22 throughFIG. 29). Slow acetylators of 3,4-diaminopyridine are more likely thanfast acetylators to have drug-related adverse events and also greaternumber of drug-related adverse events (Example 18, FIGS. 28 and 29).Fast acetylators have lower plasma levels of 3,4 DAP (FIGS. 25a, 25b,25c, and 25d, and 26a, 26b, and 26c ) and higher levels of themetabolite (FIGS. 27a, 27b, and 27c ), reduced exposure, and thepotential for lower or lack of efficacy.

Determining a patient's status as a fast or slow acetylator allows forthe optimization of exposure and efficacy and minimization of adverseevents and adds significant value for treating a LEMS patient.

EMBODIMENTS

Any of the following embodiments within embodiments may be combined withany other embodiment disclosed herein.

For the compositions and methods described herein, exemplary components,and compositional ranges thereof, can be selected from the variousexamples provided herein.

Other features and advantages of the invention will become apparent fromthe following detailed description. It should be understood, however,that the detailed description and the specific examples, whileindicating embodiments of the invention, are given by way ofillustration only, because various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

In some or any embodiments, the methods disclosed herein can bepracticed with 3,4-diaminopyridine or a pharmaceutically acceptable saltthereof or with 4-aminopyridine or a pharmaceutically acceptable saltthereof. A person of ordinary skill in the art would understand that adisease which is sensitive to treatment with 3,4-DAP will also besensitive to treatment with 4-aminopyridine.

To maximize efficacy in fast acetylators, 3,4-DAP can be taken on anempty stomach (e.g., 1 hour before or 2 hours after a meal) or the dosecan be increased and optionally taken with food at the higher dose. Toreduce the frequency and/or severity of adverse events, in slowacetylators, 3,4-DAP can be taken with food or the dose can bedecreased.

In some or any embodiments, the human is female. In some or anyembodiments, the human is female and is told to take 3,4-DAP with food.In some or any embodiments, the human is female and is told she has agreater risk of frequency and/or severity of side effects as compared toa male.

In some or any embodiments, 3,4-DAP is administered at a specified timeincluding but not limited to morning, day, night, same time of the day,and/or one or more times a day.

In some or any embodiments, the disclosed methods also comprise the stepof providing to the patient in need thereof a therapeutically effectiveamount of 3,4-diaminopyridine. The therapeutically effective amount willvary depending on the condition to be treated, and can be readilydetermined by the treating physician based on improvement in desiredclinical symptoms.

In any or all embodiments, the disease or 3,4-DAP-sensitive disease isLEMS, myasthenia gravis, multiple sclerosis, or congenital myasthenia.

In some or any embodiments, 3,4-diaminopyridine is administered as aphosphate salt. In some or any embodiments, 3,4-diaminopyridine isadministered as a tartrate salt. In some or any embodiments,3,4-diaminopyridine is administered as a free base.

Disclosed herein is a method comprising administering3,4-diaminopyridine or a pharmaceutically acceptable salt thereof, to ahuman in need thereof, and informing said human that the frequencyand/or severity of side effect(s) of said 3,4-DAP or pharmaceuticallyacceptable salt thereof are decreased when it is ingested with foodcompared to when ingested without food. In some embodiments or anyembodiments, the 3,4-DAP is administered with food and the food is ahigh-fat, high-calorie meal. In some or any embodiments, the method canbe practiced in combination with any of the methods in embodiment 1.

Embodiment 1

In some or any embodiments, the 3,4-DAP is administered shortlyfollowing food.

In some or any embodiments, the composition is administered to thepatient when the stomach is full, for example, concurrently withingestion of food, 5 min or less after ingestion of food, 10 min or lessafter ingestion of food, 30 min or less after ingestion of food, 35 minor less after ingestion of food, 40 min or less after ingestion of food,45 min or less after ingestion of food, 60 min or less after ingestionof food, 75 min or less after ingestion of food, 90 min or less afteringestion of food, 105 min or less after ingestion of food, or 120 minor less after ingestion of food. In some or any of the aboveembodiments, the composition is administered to the patient when thestomach is full, for example, concurrently with ingestion of food, 5 minor less after ingestion of food, 10 min or less after ingestion of food,30 min or less after ingestion of food, 35 min or less after ingestionof food, 40 min or less after ingestion of food, 45 min or less afteringestion of food, or 60 min or less after ingestion of food. In some orany embodiments, the composition is administered to the patient when thestomach is full, for example, concurrently with ingestion of food, 5 minor less after ingestion of food, 10 min or less after ingestion of food,30 min or less after ingestion of food, or 35 min or less afteringestion of food.

In some or any embodiments, the 3,4-DAP and the food may be ingested atapproximately the same time, or the 3,4-DAP may be ingested before orafter the food. The period of time between consuming the food and taking3,4-DAP may be about 5 min or less. For example, 3,4-DAP may beadministered before food in an interval of 30 min or less, 25 min orless, 20 min or less, 15 min or less, 10 min or less, or 5 min or less.

In some or any embodiments, the 3,4-DAP is administered with food andthe food is a high-fat, high-calorie meal.

In some or any embodiments, there is a decrease in any one, two, threeor all of the following parameters when 3,4-DAP is ingested with foodcompared to when ingested without food: mean plasma concentration,C_(max), AUC_((0-t)) and/or AUC_((0-inf)). In exemplary embodiments, thepatient is informed that administration of 3,4-diaminopyridine with ameal decreases C_(max) and AUC compared to administration of3,4-diaminopyridine without food (in a fasting condition). In someembodiments, the relative decrease in C_(max) can be at least about 50%,in another example about 40% or less, in another example by about 35% orless, in another example by about 30% or less, in another example byabout 30% or less, in another example by about 25% or less, in anotherexample by about 20% or less, in another example by about 15% or less,in another example by about 10% or less. In some embodiments, therelative decrease in AUC(0-t) can be at least about 50%, in anotherexample about 40% or less, in another example by about 35% or less, inanother example by about 30% or less, in another example by about 30% orless, in another example by about 25% or less, in another example byabout 20% or less, in another example by about 15% or less, in anotherexample by about 10% or less. In some embodiments, the relative decreasein AUC(0-inf) can be at least about 50%, in another example about 40% orless, in another example by about 35% or less, in another example byabout 30% or less, in another example by about 30% or less, in anotherexample by about 25% or less, in another example by about 20% or less,in another example by about 15% or less, in another example by about 10%or less.

In some or any embodiments, the patient is informed that ingestionshortly following a meal results in a decrease in side effect(s). Insome or any embodiments, the side effect(s) to be reduced in frequencyor severity is described in any of the embodiments or examples, forexample the following embodiments.

In some or any embodiments, the side effect(s) to be reduced infrequency or severity is independently selected from a nervous systemdisorder, a gastrointestinal disorder, general disorder, infection orinfestation, skin and subcutaneous tissue disorder, vascular disorder,cardiac disorder, and musculoskeletal and connective tissue disorder.

In some or any embodiments, the frequency of side effect(s) is reducedby about 30-35%. In some or any embodiments, the frequency of sideeffect(s) is reduced by about at least a third. In some or anyembodiments, the frequency of side effect(s) is reduced by about 25-30%.In some or any embodiments, the frequency of side effect(s) is reducedby about 20-25%. In some or any embodiments, the frequency of sideeffect(s) is reduced by about 15-20%. In some or any embodiments, thefrequency of side effect(s) is reduced by about 10-15%. In some or anyembodiments, the frequency of side effect(s) is reduced by about 5-10%.

In some or any embodiments, the side effect is a nervous systemdisorders and is decreased by about at least 30%. In some or anyembodiments, the frequency of side effects was reduced by about 25-30%.In some or any embodiments, the frequency of side effects was reduced byabout 20-25%. In some or any embodiments, the frequency of side effectswas reduced by about 15-20%. In some or any embodiments, the frequencyof side effects was reduced by about 10-15%. In some or any embodiments,the frequency of side effects was reduced by about 5-10%. In some or anyembodiments, the nervous system disorder that is decreased isparaesthesia, e.g. oral or skin.

In some or any embodiments, the side effect is paraesthesia and thefrequency is decreased by at least about 35%, in another example byabout 30-35%, in another example by about 25-30%, in another example byabout 20-25%, in another example by about 15-20%, in another example byabout 15-20%, in another example by 10-15%.

In some or any embodiments, the side effect is dizziness and thefrequency is decreased by at least about 60%, in another example byabout 55-60%, in another example by about 50-55%, in another example byabout 45-50%, in another example by about 40-45%, in another example byabout 35-40%, in another example by 30-35%, in another example by about25-30%, in another example by about 20-25%, in another example by about15-20%, in another example by about 10-15%, in another example by about5-10%.

In some or any embodiments, the side effect is oral headache and thefrequency is decreased by at least about 75%, in another example byabout 70-75%, in another example by about 65-70%, in another example byabout 60-65%, in another example by about 55-60%, in another example byabout 50-55%, in another example by about 45-50%, in another example byabout 40-45%, in another example by about 35-40%, in another example by30-35%, in another example by about 25-30%, in another example by about20-25%, in another example by about 15-20%, in another example by about10-15%, in another example by about 5-10%.

In some or any embodiments, the side effect is hypoaesthesia and thefrequency is decreased by about 100%, in another example by about90-100%, in another example by about 80-90%, in another example by about70-80%, in another example by about 60-70%, in another example by about55-60%, in another example by about 50-55%, in another example by about45-50%, in another example by about 40-45%, in another example by about35-40%, in another example by 30-35%, in another example by about25-30%, in another example by about 20-25%, in another example by about15-20%, in another example by about 10-15%, in another example by about5-10%.

In some or any embodiments, the side effect is facial hypoaesthesia andthe frequency is decreased by about 100%, in another example by about90-100%, in another example by about 80-90%, in another example by about70-80%, in another example by about 60-70%, in another example by about55-60%, in another example by about 50-55%, in another example by about45-50%, in another example by about 40-45%, in another example by about35-40%, in another example by 30-35%, in another example by about25-30%, in another example by about 20-25%, in another example by about15-20%, in another example by about 10-15%, in another example by about5-10%.

In some or any embodiments, the side effect is a gastrointestinaldisorder and is decreased by at least about 80% amount, in anotherexample by about 70-80%, in another example by about 60-70%, in anotherexample by about 55-60%, in another example by about 50-55%, in anotherexample by about 45-50%, in another example by about 40-45%, in anotherexample by about 35-40%, in another example by 30-35%, in anotherexample by about 25-30%, in another example by about 20-25%, in anotherexample by about 15-20%, in another example by about 10-15%, in anotherexample by about 5-10%.

In some or any embodiments, the side effect is a nausea and is decreasedby at least about 65% amount, in another example by about two thirds, inanother example by about 60-65%, in another example by about 55-60%, inanother example by about 50-55%, in another example by about 45-50%, inanother example by about 40-45%, in another example by about 35-40%, inanother example by 30-35%, in another example by about 25-30%, inanother example by about 20-25%, in another example by about 15-20%, inanother example by about 10-15%, in another example by about 5-10%.

In some or any embodiments, the side effect is abdominal pain and/ortenderness and the frequency is decreased by about 100%, in anotherexample by about 90-100%, in another example by about 80-90%, in anotherexample by about 70-80%, in another example by about 60-70%, in anotherexample by about 55-60%, in another example by about 50-55%, in anotherexample by about 45-50%, in another example by about 40-45%, in anotherexample by about 35-40%, in another example by 30-35%, in anotherexample by about 25-30%, in another example by about 20-25%, in anotherexample by about 15-20%, in another example by about 10-15%, in anotherexample by about 5-10%.

In some or any embodiments, the side effect is diarrhea and thefrequency is decreased by about 100%, in another example by about90-100%, in another example by about 80-90%, in another example by about70-80%, in another example by about 60-70%, in another example by about55-60%, in another example by about 50-55%, in another example by about45-50%, in another example by about 40-45%, in another example by about35-40%, in another example by 30-35%, in another example by about25-30%, in another example by about 20-25%, in another example by about15-20%, in another example by about 10-15%, in another example by about5-10%.

Phenotyping Methods

Caffeine methods: In some or any embodiments, a subject is determined tobe a slow or fast acetylator by administering caffeine to the subject,taking a urine sample, and measuring the metabolites of caffeine(“caffeine test”). In some or any embodiments of the caffeine test, 150mg caffeine is administered. In some or any embodiments of the caffeinetest, the following ratio is determined: (AFMU+AAMU)/(AFMU+AAMU+1X+1U),wherein AFMU is the concentration of5-acetylamino-6-formylamino-3-methyluracil, AAMU is the concentration of5-acetylamino-6-amino-3-methyluracil, 1X is the concentration of1-methylxanthine; and 1U is the concentration of 1 methylurate. In someor any embodiments of the caffeine test, a fast acetylator has a ratioof greater than about 0.2, in another example between about 0.2 to about0.3, and a slow acetylator has a ratio of about 0.2 or less, for examplebetween about 0.1 and about 0.2 (inclusive). In some or any embodiments,the caffeine test is administered substantially as described in Example15a.

In some or any embodiments, a subject's phenotype is determinedsubstantially as described in Example 15, including any of the citedreferences, the disclosure of each of which is herein incorporated byreference in its entirety.

In some or any embodiments, a subject's NAT polymorphism phenotype isdetermined using the caffeine test as described in any embodiments orexamples herein.

3,4-DAP Test: In some or any embodiments, the subject is determined tobe a slow or fast acetylator by administering 3,4-DAP, or apharmaceutically acceptable salt thereof, to the subject and measuringthe amount of 3,4-DAP and N-(4-aminopyridin-3-yl)acetamide. In some orany embodiments, the fast acetylator has an AUC_(0-inf) ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of greater than about 20. Insome or any embodiments, a slow acetylator has a ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of less than about 20. In someor any embodiments, the fast acetylator has an AUC_(0-inf) ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of greater than about 25. Insome or any embodiments, a slow acetylator has a ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of less than about 15. In someor any embodiments, the fast acetylator has an AUC_(0-inf) ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of greater than about 30. Insome or any embodiments, a slow acetylator has a ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of less than about 10. In someor any embodiments, the fast acetylator has an AUC_(0-inf) ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of greater than about 30 whendosed with a single dose of 5-30 mg equivalent of free base. In some orany embodiments, a slow acetylator has a ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of less than about 10 whendosed with a single dose of 5-30 mg equivalent of free base.

Genotyping Methods

NAT Genotyping Methods: In some or any embodiments, a subject's NATpolymorphism genotype is determined by screening a subject's NAT2 geneusing one or both of the following molecular probes: C282T and T341C. Insome or any embodiments, the subject's NAT1 gene is examined for one ofthe following seven SNPs *3, *10, *11, *14, *15, *17. In some or anyembodiments, the subject's NAT2 gene is examined for one of thefollowing four SNPs *5, *6, *7, and *14. In some or any embodiments, asubject with no fast alleles is a slow metabolizer. In some or anyembodiments, the subject whose genotype includes at least one fastallele is a fast acetylator. In some or any embodiments, the subjectwhose genotype includes two fast alleles is a fast acetylator. In someor any embodiments, a subject is genotyped substantially according toTable 4. In some or any embodiments, a subject's genotype is determinedsubstantially as described in Example 16.

Embodiment 2

Disclosed herein is a method of treating comprising determining whethera subject is a slow acetylator or fast acetylator; selecting a dose of3,4-DAP or a pharmaceutically acceptable salt thereof; and administeringthe dose of 3,4-DAP to the subject in need thereof. In some or any ofthe above embodiments in embodiment 2, the method further comprisesinforming said human who is a slow acetylator to take said 3,4-DAP orpharmaceutically acceptable salt thereof with food or informing saidhuman who is a fast acetylator to take said 3,4-DAP or pharmaceuticallyacceptable salt thereof without food. In some or any of the aboveembodiments in embodiment 2, the method further comprises informing saidhuman who is a slow acetylator that the frequency and/or severity ofside effect(s), as described in any of the embodiments or examplesherein, of said 3,4-DAP or pharmaceutically acceptable salt thereof aredecreased when it is ingested with food compared to when ingestedwithout food; or informing said human who is a fast acetylator that theefficacy of said 3,4-DAP or pharmaceutically acceptable salt thereof isincreased when it is ingested without food compared to when ingestedwith food. In some or any of the above embodiments in embodiment 2, asubject is determined to be a slow or fast acetylator by performing anyof the phenotyping methods as described by any of the embodiments orexamples herein. In some or any of the above embodiments in embodiment2, a subject is determined to be a slow or fast acetylator bydetermining a subject's genotype as described by any of the embodimentsor examples herein. In some or any embodiments in embodiment 2, asubject is determined to be a slow or fast acetylator by administeringcaffeine to the subject and measuring the metabolites of caffeine. Insome or any embodiments in embodiment 2, 150 mg caffeine isadministered. In some or any embodiments in embodiment 2, the followingratio is determined: (AFMU+AAMU)/(AFMU+AAMU+1X+1U), wherein AFMU is theconcentration of 5-acetylamino-6-formylamino-3-methyluracil, AAMU is theconcentration of 5-acetylamino-6-amino-3-methyluracil, 1X is theconcentration of 1-methylxanthine; and 1U is the concentration of 1methylurate. In some or any embodiments in embodiment 2, a fastacetylator has a ratio of greater than about 0.2, in another examplebetween about 0.2 to about 0.3, and a slow acetylator has a ratio ofabout 0.2 or less, for example between about 0.1 and about 0.2(inclusive).

Embodiment 3

Disclosed herein is a method comprising a) determining a subject's NATpolymorphism phenotype; b) selecting a dose of 3,4-DAP or apharmaceutically acceptable salt thereof based on the subject'sacetylator status; and c) administering the dose of 3,4-DAP to a subjectin need thereof. In some or any of the above embodiments in embodiment3, the method further comprises informing said human whose phenotype isslow acetylation to take said 3,4-DAP or pharmaceutically acceptablesalt thereof with or informing said human whose phenotype is fastacetylation to take said 3,4-DAP or pharmaceutically acceptable saltthereof without food. In some or any of the above embodiments inembodiment 3, the method further comprises informing the subject 1)whose phenotype is slow acetylation that the frequency and/or severityof side effect(s) of said 3,4-DAP or pharmaceutically acceptable saltthereof are decreased when it is ingested with food compared to wheningested without food; or 2) whose phenotype is fast acetylation thatthe efficacy of said 3,4-DAP or pharmaceutically acceptable salt thereofis increased when it is ingested without food compared to when ingestedwith food. In some or any of the above embodiments in embodiment 3, asubject's phenotype is determined by performing any of the phenotypingmethods as described by any of the embodiments or examples herein. Insome or any embodiments in embodiment 3, a subject's phenotype isdetermined by administering caffeine to the subject and measuring themetabolites of caffeine. In some or any embodiments in embodiment 3, 150mg caffeine is administered. In some or any embodiments in embodiment 3,the following ratio is determined: (AFMU+AAMU)/(AFMU+AAMU+1X+1U),wherein AFMU is the concentration of5-acetylamino-6-formylamino-3-methyluracil, AAMU is the concentration of5-acetylamino-6-amino-3-methyluracil, 1X is the concentration of1-methylxanthine; and 1U is the concentration of 1 methylurate. In someor any embodiments in embodiment 3, a fast acetylator has a ratio ofgreater than about 0.2, in another example between about 0.2 to about0.3, in another example greater than or equal to about 0.3, and a slowacetylator has a ratio of about 0.2 or less, for example between about0.1 and about 0.2 (inclusive).

Embodiment 4

Disclosed herein is a method comprising a) determining a subject's NAT2polymorphism genotype; b) selecting a dose of 3,4-DAP or apharmaceutically acceptable salt thereof based on the number of fastalleles the subject has; and c) administering the dose of 3,4-DAP to asubject in need thereof. In some embodiments or any of the aboveembodiments in embodiment 4, the method further comprises informing saidhuman to take said 3,4-DAP or pharmaceutically acceptable salt thereofwithout food if the subject has two fast alleles or with food if thesubject has no fast alleles. In some embodiments or any of the aboveembodiments in embodiment 4, the method further comprises informing thesubject a) who has no fast alleles that the frequency and/or severity ofside effect(s) of said 3,4-DAP or pharmaceutically acceptable saltthereof are decreased when it is ingested with food compared to wheningested without food; or b) who has two fast alleles that the efficacyof said 3,4-DAP or pharmaceutically acceptable salt thereof is increasedwhen it is ingested without food compared to when ingested with food. Insome embodiments or any of the above embodiments in embodiment 4, asubject's NAT2 genotype is determined using a method as described in anyof the embodiments or examples herein. In some embodiments or anyembodiments in embodiment 4, a subject's NAT2 gene is screened formutations using one or both of the following molecular probes: C282T andT341C. In some embodiments or any embodiments in embodiment 4, thesubject's NAT1 gene is examined for one of the following seven SNPs *3,*10, *11, *14, *15, *17. In some embodiments or any embodiments inembodiment 4, the subject's NAT2 gene is examined for one of thefollowing four SNPs *5, *6, *7, and *14. In some embodiments or anyembodiments in embodiment 4, a subject is genotyped substantiallyaccording to Table 4.

Embodiment 5

Disclosed herein is a method comprising selecting a dose of 3,4-DAP or apharmaceutically acceptable salt thereof for a subject who is a fastacetylator; and administering the dose of 3,4-DAP to the subject in needthereof without food. In another embodiment, the dose is selectedaccording to any of the embodiments and examples as described herein.

Embodiment 6

Disclosed herein is a method comprising selecting a dose of 3,4-DAP or apharmaceutically acceptable salt thereof for a subject who is a slowacetylator; administering the dose of 3,4-DAP to the subject in needthereof with food. In another embodiment, the dose is selected accordingto any of the embodiment and examples as disclosed herein.

Embodiment 7

Disclosed herein is a method comprising determining whether a subject isa slow acetylator or fast acetylator; and administering3,4-diaminopyridine (3,4-DAP), comprising administering to a human inneed thereof 3,4-DAP or a pharmaceutically acceptable salt thereof in anamount that is adjusted to the subject's acetylator status. In some orany of the above embodiments in embodiment 7, the subject is determinedto be a slow or fast acetylator by performing a phenotyping method asdescribed by any of the embodiments or examples herein. In some or anyembodiments in embodiment 7, the subject is determined to be a slow orfast acetylator by administering caffeine to the subject and measuringthe metabolites of caffeine. In some or any embodiments in embodiment 7,150 mg caffeine is administered. In some or any embodiments inembodiment 7, the following ratio is determined:(AFMU+AAMU)/(AFMU+AAMU+1X+1U), wherein AFMU is the concentration of5-acetylamino-6-formylamino-3-methyluracil, AAMU is the concentration of5-acetylamino-6-amino-3-methyluracil, 1X is the concentration of1-methylxanthine; and 1U is the concentration of 1 methylurate. In someor any embodiments in embodiment 7, a fast acetylator has a ratio ofgreater than about 0.2, in another example between about 0.2 to about0.3, in another example greater than or equal to about 0.3, and a slowacetylator has a ratio of about 0.2 or less, for example between about0.1 and about 0.2 (inclusive).

Embodiment 8

Disclosed herein is a method comprising selecting a dose of 3,4-DAP or apharmaceutically acceptable salt thereof for a subject who is a fastacetylator; and administering the dose of 3,4-DAP to the subject in needthereof without food. In another embodiment, the dose is selectedaccording to any of the embodiments and examples as described herein.

Embodiment 9

Disclosed herein is a method comprising determining whether a subject isa slow acetylator or fast acetylator; selecting a dose of 3,4-DAP or apharmaceutically acceptable salt thereof in an amount that is adjustedto the subject's acetylator status; and administering the dose of3,4-DAP to the subject in need thereof. In some or any of the aboveembodiments in embodiment 9, the subject is determined to be a slow orfast acetylator by performing a phenotyping method as described by anyof the embodiments or examples herein. In some or any embodiments inembodiment 9, the subject is determined to be a slow or fast acetylatorby administering caffeine to the subject and measuring the metabolitesof caffeine. In some or any embodiments in embodiment 9, 150 mg caffeineis administered. In some or any embodiments in embodiment 9, thefollowing ratio is determined: (AFMU+AAMU)/(AFMU+AAMU+1X+1U), whereinAFMU is the concentration of 5-acetylamino-6-formylamino-3-methyluracil,AAMU is the concentration of 5-acetylamino-6-amino-3-methyluracil, 1X isthe concentration of 1-methylxanthine; and 1U is the concentration of 1methylurate. In some or any embodiments in embodiment 9, a fastacetylator has a ratio of greater than about 0.2, in anther examplebetween about 0.2 to about 0.3, in another example greater than or equalto about 0.3, and a slow acetylator has a ratio of about 0.2 or less,for example between about 0.1 and about 0.2 (inclusive).

Embodiment 10

Disclosed herein is a method of determining whether a subject who has a3,4-DAP-sensitive disease is a slow or fast acetylator. In some or anyembodiments of embodiment 10, the subject is determined to be a slow orfast acetylator by performing a phenotyping method as described by anyof the embodiments or examples herein. In some or any embodiments ofembodiment 10, the subject is determined to be a slow or fast acetylatorby administering caffeine to the subject and measuring the metabolitesof caffeine. In some or any embodiments, the slow acetylator has a ratioof caffeine metabolites of about 0.2 or less as calculated with thefollowing formula: (AFMU+AAMU)/(AFMU+AAMU+1X+1U), wherein AFMU is theconcentration of 5-acetylamino-6-formylamino-3-methyluracil, AAMU is theconcentration of 5-acetylamino-6-amino-3-methyluracil, 1X is theconcentration of 1-methylxanthine; and 1U is the concentration of 1methylurate. In some or any embodiments, the fast acetylator has a ratioof caffeine metabolites of greater than about 0.2, in another examplebetween about 0.2 to about 0.3, in another example greater than or equalto about 0.3, as calculated with the following formula:(AFMU+AAMU)/(AFMU+AAMU+1X+1U). In some or any embodiments of embodiment10, the subject is determined to be a slow or fast acetylator bydetermining the subject's NAT polymorphism genotype using methods asdescribed in any of the embodiments or examples herein. In some or anyembodiments, the fast acetylator has two fast alleles. In some of anyembodiments, the slow acetylator has no fast alleles. In some or anyembodiments, a subject's NAT2 gene is screened for mutations using oneor both of the following molecular probes: C282T and T341C.

Embodiment 11

Disclosed herein is a method of treating a 3,4-DAP-sensitive diseasecomprising determining whether a subject is a slow acetylator or fastacetylator; selecting a dose of 3,4-DAP or a pharmaceutically acceptablesalt thereof in an amount that is adjusted to the subject's acetylatorstatus; and administering the dose of 3,4-DAP to the subject in needthereof.

Embodiment A

Provided herein is a method of determining whether a subject who has a3,4-DAP-sensitive disease is a slow or fast acetylator. In some or anyembodiments, the subject is determined to be a slow or fast acetylatorby determining a subject's phenotype using one of the phenotypingmethods as described in any of the embodiments or examples hereinincluding any of the references which are herein are incorporated byreference, in another example by administering the caffeine test asdescribed by any of the embodiments or examples herein, for example inExample 15a. In some or any embodiments, the subject is determined to bea slow or fast acetylator by administering caffeine to the subject andmeasuring the metabolites of caffeine. In some or any embodiments, theslow acetylator has a ratio of caffeine metabolites of about 0.2 or lessas calculated with the following formula: (AFMU+AAMU)/(AFMU+AAMU+1X+1U),wherein AFMU is the concentration of5-acetylamino-6-formylamino-3-methyluracil, AAMU is the concentration of5-acetylamino-6-amino-3-methyluracil, 1X is the concentration of1-methylxanthine; and 1U is the concentration of 1 methylurate. In someor any embodiments, the fast acetylator has a ratio of caffeinemetabolites of greater than about 0.2 as calculated with the followingformula: (AFMU+AAMU)/(AFMU+AAMU+1X+1U), wherein AFMU is theconcentration of 5-acetylamino-6-formylamino-3-methyluracil, AAMU is theconcentration of 5-acetylamino-6-amino-3-methyluracil, 1X is theconcentration of 1-methylxanthine; and 1U is the concentration of 1methylurate. In some or any embodiments, the subject is determined to bea slow or fast acetylator by determining the subject's NAT polymorphismgenotype using any one of the genotyping methods as described in any ofthe embodiments and examples including any references which are hereinare incorporated by reference. In some or any embodiments, the subject'sgenotype is determined using one or both of the molecular probes C282Tand T341C. In some or any embodiments, the subject whose genotypeincludes no fast alleles is a slow acetylator. In some or anyembodiments, the subject whose genotype includes at least one fastallele is a fast acetylator. In some or any embodiments, the subjectwhose genotype includes two fast alleles is a fast acetylator. In someor any embodiments, the subject is determined to be a slow or fastacetylator by administering the 3,4-DAP test. In some or anyembodiments, the subject is determined to be a slow or fast acetylatorby administering 3,4-DAP, or a pharmaceutically acceptable salt thereof,to the subject and measuring the amount of 3,4-DAP andN-(4-aminopyridin-3-yl)acetamide. In some or any embodiments, the fastacetylator has an AUC_(0-inf) ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of greater than about 20. Insome or any embodiments, a slow acetylator has a ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of less than about 20. In someor any embodiments, the fast acetylator has an AUC_(0-inf) ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of greater than about 25. Insome or any embodiments, a slow acetylator has a ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of less than about 15. In someor any embodiments, the fast acetylator has an AUC_(0-inf) ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of greater than about 30. Insome or any embodiments, a slow acetylator has a ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of less than about 10. In someor any embodiments, the fast acetylator has an AUC_(0-inf) ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of greater than about 30 whendosed with a single dose of 5-30 mg equivalent of free base. In some orany embodiments, a slow acetylator has a ratio ofN-(4-aminopyridin-3-yl)acetamide/3,4-DAP of less than about 10 whendosed with a single dose of 5-30 mg equivalent of free base.

Embodiment B

Disclosed herein is a method which comprises selecting a dose of 3,4-DAPor a pharmaceutically acceptable salt thereof in an amount that isadjusted to the subject's acetylator status which method optionallyfurther comprises any of the embodiments in Embodiment A. In some or anyembodiments, for example any of embodiment A, the dose is selected toachieve C_(max) (maximum plasma concentration) of 3,4-DAP of about 15ng/mL or less about 20 ng/mL or less, about 25 ng/mL or less, about 30ng/mL or less, about 35 ng/mL or less, about 40 ng/mL or less, about 45ng/mL or less, about 50 ng/mL or less, about 55 ng/mL or less, about 60ng/mL or less, about 65 ng/mL or less, about 70 ng/mL or less, about 75ng/mL or less, about 80 ng/mL or less, about 85 ng/mL or less, or about90 ng/mL or less. In some or any embodiments, for example any ofembodiment A, the dose is selected to achieve C_(max) of 3,4-DAP ofabout 25 ng/mL or less, about 30 ng/mL or less, about 35 ng/mL or less,about 40 ng/mL or less, about 45 ng/mL or less, about 50 ng/mL or less,about 55 ng/mL or less, or about 60 ng/mL or less. In some or anyembodiments, for example any of embodiment A, the dose is selected toachieve C_(max) of 3,4-DAP of about 40 ng/mL or less, about 45 ng/mL orless, about 50 ng/mL or less, or about 55 ng/mL or less. In some or anyembodiments, for example any of embodiment A, the dose is selected toachieve C_(max) of 3,4-DAP of less than or equal to about 50 ng/mL andgreater than or equal to about 10 ng/mL. In some or any embodiments, forexample any of embodiment A, the dose is selected to achieve C_(max) of3,4-DAP of less than or equal to about 50 ng/mL and greater than orequal to about 15 ng/mL. In some or any embodiments, for example any ofembodiment A, the dose is selected to achieve C_(max) of 3,4-DAP of lessthan or equal to about 50 ng/mL and greater than or equal to about 20ng/mL. In some or any embodiments, for example any of embodiment A, thedose is selected to achieve C_(max) of 3,4-DAP of less than or equal toabout 40 ng/mL and greater than or equal to about 10 ng/mL. In some orany embodiments, for example any of embodiment A, the dose is selectedto achieve C_(max) of 3,4-DAP of less than or equal to about 40 ng/mLand greater than or equal to about 15 ng/mL. In some or any embodiments,for example any of embodiment A, the dose is selected to achieve C_(max)of 3,4-DAP of less than or equal to about 40 ng/mL and greater than orequal to about 20 ng/mL. In some or any embodiments, for example any ofembodiment A, the dose is selected to achieve C_(max) of 3,4-DAP of lessthan or equal to about 35 ng/mL and greater than or equal to about 25ng/mL. In some or any embodiments, for example any of embodiment A, thedose is selected to achieve C_(max) of 3,4-DAP of about 30 ng/mL. Inanother embodiment, any embodiments A can be practiced with any ofembodiments B.

Embodiment C

In some or any embodiments, disclosed herein is a method which comprisesadministering the dose of 3,4-DAP or a pharmaceutically acceptable saltthereof which method optionally further comprises any of the embodimentsin embodiment A and/or B. In some or any embodiments, for example any ofembodiment A and/or B, the dose is administered to a slow acetylatorwith food and to a fast acetylator without food. In another embodiment,any embodiment of embodiments A and/or B can be practiced with any ofembodiments C.

Embodiment D

In some or any embodiments, disclosed herein is a method which comprisesinforming said subject who is a slow acetylator to take said 3,4-DAP ora pharmaceutically acceptable salt thereof with food or informing saidsubject who is a fast acetylator to take said 3,4-DAP orpharmaceutically acceptable salt thereof without food which methodoptionally further comprises any of the embodiments in embodiment A, B,and/or C. In some or any embodiments, for example any of embodiment A, Band/or C, the method comprises informing said subject who is a slowacetylator that the frequency and/or severity of side effect(s) of said3,4-DAP or pharmaceutically acceptable salt thereof are decreased whenit is ingested with food compared to when ingested without food; orinforming said subject who is a fast acetylator that efficacy of said3,4-DAP or pharmaceutically acceptable salt thereof is increased when itis ingested without food compared to when ingested with food. In anotherembodiment, any embodiment of embodiments A, B, and/or C can bepracticed with any of embodiments D.

Embodiment E

In some or any embodiments, for example any of embodiment C and/or D,the food is a high-fat, high-calorie meal. In another embodiment, anyembodiment of embodiments C and/or D can be practiced with any ofembodiments E.

Embodiment F

In some or any embodiments, for example any of embodiment D and/or E,the slow acetylator is informed that the frequency of side effect(s) isdecreased by about 30% when ingested with food. In another embodiment,any embodiment of embodiments D and/or E can be practiced with any ofembodiments F.

Embodiment G

In some or any embodiments, for example any of embodiment D, E and/or F,at least one side effect is a nervous system disorder. In some or anyembodiments, for example any of embodiment D, E and/or F, at least oneside effect is gastrointestinal disorder. In another embodiment, anyembodiment of embodiments D, E, and/or F can be practiced with any ofembodiments G.

Embodiment H

Provided herein is a method of selecting a dose of 3,4-DAP or apharmaceutically acceptable salt thereof in an amount that is adjustedto the subject's acetylator status. In some or any embodiments, the doseis selected to achieve C_(max) of 3,4-DAP of about 15 ng/mL or lessabout 20 ng/mL or less, about 25 ng/mL or less, about 30 ng/mL or less,about 35 ng/mL or less, about 40 ng/mL or less, about 45 ng/mL or less,about 50 ng/mL or less, about 55 ng/mL or less, about 60 ng/nL or less,about 65 ng/nL or less, about 70 ng/mL or less, about 75 ng/mL or less,about 80 ng/mL or less, about 85 ng/mL or less, or about 90 ng/mL orless. In some or any embodiments, the dose is selected to achieveC_(max) of 3,4-DAP of about 25 ng/mL or less, about 30 ng/mL or less,about 35 ng/mL or less, about 40 ng/mL or less, about 45 ng/mL or less,about 50 ng/mL or less, about 55 ng/mL or less, or about 60 ng/mL orless. In some or any embodiments, the dose is selected to achieveC_(max) of 3,4-DAP of about 40 ng/mL or less, about 45 ng/mL or less,about 50 ng/mL or less, or about 55 ng/mL or less. In some or anyembodiments, the dose is selected to achieve C_(max) of 3,4-DAP of lessthan or equal to about 50 ng/mL and greater than or equal to about 10ng/mL. In some or any embodiments, the dose is selected to achieveC_(max) of 3,4-DAP of less than or equal to about 50 ng/mL and greaterthan or equal to about 15 ng/mL. In some or any embodiments, the dose isselected to achieve C_(max) of 3,4-DAP of less than or equal to about 50ng/mL and greater than or equal to about 20 ng/mL. In some or anyembodiments, the dose is selected to achieve C_(max) of 3,4-DAP of lessthan or equal to about 40 ng/mL and greater than or equal to about 10ng/mL. In some or any embodiments, the dose is selected to achieveC_(max) of 3,4-DAP of less than or equal to about 40 ng/mL and greaterthan or equal to about 15 ng/mL. In some or any embodiments, the dose isselected to achieve C_(max) of 3,4-DAP of less than or equal to about 40ng/mL and greater than or equal to about 20 ng/mL. In some or anyembodiments, the dose is selected to achieve C_(max) of 3,4-DAP of lessthan or equal to about 35 ng/mL and greater than or equal to about 25ng/mL. In some or any embodiments, the dose is selected to achieveC_(max) of 3,4-DAP of about 30 ng/mL. In some or any embodiments, themethod further comprises administering the dose of 3,4-DAP or apharmaceutically acceptable salt thereof. In some or any embodiments,the dose is administered to a slow acetylator with food and to a fastacetylator without food. In some or any embodiments, the food is ahigh-fat, high-calorie meal. In some or any embodiments, the subject isfemale.

Embodiment J

Provided herein is a method of administering a dose of 3,4-DAP or apharmaceutically acceptable salt thereof in an amount that is adjustedto the subject's acetylator status. In some or any embodiments, the doseis selected to achieve C_(max) of 3,4-DAP of about 15 ng/mL or lessabout 20 ng/mL or less, about 25 ng/mL or less, about 30 ng/mL or less,about 35 ng/mL or less, about 40 ng/mL or less, about 45 ng/mL or less,about 50 ng/mL or less, about 55 ng/mL or less, about 60 ng/mL or less,about 65 ng/mL or less, about 70 ng/mL or less, about 75 ng/mL or less,about 80 ng/mL or less, about 85 ng/mL or less, or about 90 ng/mL orless. In some or any embodiments, the dose is selected to achieveC_(max) of 3,4-DAP of about 25 ng/mL or less, about 30 ng/mL or less,about 35 ng/mL or less, about 40 ng/mL or less, about 45 ng/mL or less,about 50 ng/mL or less, about 55 ng/mL or less, or about 60 ng/mL orless. In some or any embodiments, the dose is selected to achieveC_(max) of 3,4-DAP of about 40 ng/mL or less, about 45 ng/mL or less,about 50 ng/mL or less, or about 55 ng/mL or less. In some or anyembodiments, the dose is selected to achieve C_(max) of 3,4-DAP of lessthan or equal to about 50 ng/mL and greater than or equal to about 10ng/mL. In some or any embodiments, the dose is selected to achieveC_(max) of 3,4-DAP of less than or equal to about 50 ng/mL and greaterthan or equal to about 15 ng/mL. In some or any embodiments, the dose isselected to achieve C_(max) of 3,4-DAP of less than or equal to about 50ng/mL and greater than or equal to about 20 ng/mL. In some or anyembodiments, the dose is selected to achieve C_(max) of 3,4-DAP of lessthan or equal to about 40 ng/nL and greater than or equal to about 10ng/mL. In some or any embodiments, the dose is selected to achieveC_(max) of 3,4-DAP of less than or equal to about 40 ng/mL and greaterthan or equal to about 15 ng/mL. In some or any embodiments, the dose isselected to achieve C_(max) of 3,4-DAP of less than or equal to about 40ng/mL and greater than or equal to about 20 ng/mL. In some or anyembodiments, the dose is selected to achieve C_(max) of 3,4-DAP of lessthan or equal to about 35 ng/mL and greater than or equal to about 25ng/mL. In some or any embodiments, the dose is selected to achieveC_(max) of 3,4-DAP of about 30 ng/mL. In some or any embodiments, thedose is administered to a slow acetylator with food. In some or anyembodiments, the food is a high-fat, high-calorie meal. In some or anyembodiments, the dose is administered to a fast acetylator without food.In some or any embodiments, the subject is female.

Embodiment K

Provided herein is a method of selecting a dose of 3,4-DAP or apharmaceutically acceptable salt thereof for a subject who is a fastacetylator and informing the fast acetylator to take said 3,4-DAPwithout food. In another embodiment, the dose is selected according toany of the embodiments and examples disclosed herein.

Embodiment L

Provided herein is a method of treating a 3,4-DAP-sensitive diseasecomprising determining whether a subject is a slow acetylator or fastacetylator; selecting a dose of 3,4-DAP or a pharmaceutically acceptablesalt thereof based on the subject's acetylator status; and administeringthe dose of 3,4-DAP to the subject in need thereof. In anotherembodiment, the acetylation status, the selected dose, and theadministration, for example with or without food, is according to any ofthe embodiments and examples disclosed herein.

Embodiment M

Provided herein is a method of treating a 3,4-DAP-sensitive diseasecomprising selecting a dose of 3,4-DAP or a pharmaceutically acceptablesalt thereof in an amount adjusted to the subject's acetylator status;and administering the dose of 3,4-DAP to the subject in need thereof. Inanother embodiment, the selected dose and the administration, forexample with or without food, is according to any of the embodiments andexamples disclosed herein.

Embodiment N

Provided herein is a method of treating a 3,4-DAP-sensitive diseasecomprising administering a dose of 3,4-DAP to the subject in needthereof in an amount adjusted to the subject's acetylator status. Inanother embodiment, the administration, for example with or withoutfood, is according to any of the embodiments and examples disclosedherein.

Embodiment P

Provided herein is a method of treating a 3,4-DAP-responsive disorderwith 3,4-DAP or a pharmaceutically acceptable salt thereof in a subjectwho is a fast acetylator in a dose of about 80 mg or more of free base(regardless of whether the dose is administered as the free base or as apharmaceutically acceptable salt) per day. In some or any embodiments,the dose is about 100 mg or more of free base (regardless of whether thedose is administered as the free base or as a pharmaceuticallyacceptable salt) per day. In another embodiment, the dose isadministered with food. In another embodiment, the dose is administeredwithout food.

Embodiment Q

Provided herein is a method of treating a 3,4-DAP-responsive disorderwith 3,4-DAP or a pharmaceutically acceptable salt thereof incombination with an inhibitor of an NAT enzyme. In some or anyembodiments, the NAT inhibitor is acetaminophen, curcumin, or caffeicacid. In some or any embodiments, the NAT inhibitor is a NAT1 inhibitorand the NAT1 inhibitor is caffeic acid. In some or any embodiments, theNAT inhibitor is a NAT2 inhibitor and the NAT2 inhibitor isacetaminophen or curcumin.

Embodiments for a Slow Acetylator

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the 3,4-DAP is administeredwith food and the food is a high-fat, high-caloric meal.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the 3,4-DAP is administeredshortly following food.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the 3,4-DAP is administeredwhen the stomach is full, for example, concurrently with ingestion offood, 5 min or less after ingestion of food, 10 min or less afteringestion of food, 30 min or less after ingestion of food, 35 min orless after ingestion of food, 40 min or less after ingestion of food, 45min or less after ingestion of food, 60 min or less after ingestion offood, 75 min or less after ingestion of food, 90 min or less afteringestion of food, 105 min or less after ingestion of food, or 120 minor less after ingestion of food. In some or any of embodiments 2-4, 6,7, and 9-11, C-J, L-N, and Q, the composition is administered to thepatient when the stomach is full, for example, concurrently withingestion of food, 5 min or less after ingestion of food, 10 min or lessafter ingestion of food, 30 min or less after ingestion of food, 35 minor less after ingestion of food, 40 min or less after ingestion of food,45 min or less after ingestion of food, or 60 min or less afteringestion of food. In some or any of embodiments, for example 2-4, 6, 7,and 9-11, C-J, L-N, and Q, the composition is administered to thepatient when the stomach is full, for example, concurrently withingestion of food, 5 min or less after ingestion of food, 10 min or lessafter ingestion of food, 30 min or less after ingestion of food, or 35min or less after ingestion of food.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the 3,4-DAP is administeredwith food and the food may be ingested at approximately the same time,or the 3,4-DAP may be ingested before or after the food. The period oftime between consuming the food and taking 3,4-DAP, either swallowed ordissolved, may be about 5 min or less. For example, 3,4-DAP may beadministered 30 min, 25 min, 20 min, 15 min, 10 min, or 5 min before orafter a meal.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the frequency of sideeffect(s) was reduced by about 30-35%. In some or any of embodiments,for example 2-4, 6, 7, 9-11, C-J, L-N, and Q, the human is a slowacetylator and the frequency of side effect(s) was reduced by about atleast a third. In some or any of embodiments, for example 2-4, 6, 7,9-11, C-J, L-N, and Q, the human is a slow acetylator and the frequencyof side effect(s) was reduced by about 25-30%. In some or any ofembodiments, for example 2-4, 6, 7, 9-11, C-J, L-N, and Q, the human isa slow acetylator and the frequency of side effect(s) was reduced byabout 20-25%. In some or any of embodiments, for example 2-4, 6, 7,9-11, C-J, L-N, and Q, the human is a slow acetylator and the frequencyof side effect(s) was reduced by about 15-20%. In some or any ofembodiments, for example 2-4, 6, 7, 9-11, C-J, L-N, and Q, the human isa slow acetylator and the frequency of side effect(s) was reduced byabout 10-15%. In some or any of embodiments, for example 2-4, 6, 7,9-11, C-J, L-N, and Q, the human is a slow acetylator and the frequencyof side effect(s) was reduced by about 5-10%.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the side effect is a nervoussystem disorders and is decreased by about at least 30%. In some or anyof embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N, and Q, the humanis a slow acetylator and the frequency of side effects was reduced byabout 25-30%. In some or any of embodiments, for example 2-4, 6, 7,9-11, C-J, L-N, and Q, the human is a slow acetylator and the frequencyof side effects was reduced by about 20-25%. In some or any ofembodiments, for example 2-4, 6, 7, 9-11, C-J, L-N, and Q, the human isa slow acetylator and the frequency of side effects was reduced by about15-20%. In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J,L-N, and Q, the human is a slow acetylator and the frequency of sideeffects was reduced by about 10-15%. In some or any of embodiments, forexample 2-4, 6, 7, 9-11, C-J, L-N, and Q, the human is a slow acetylatorand the frequency of side effects was reduced by about 5-10%. In some orany of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N, and Q, thehuman is a slow acetylator and the nervous system disorder that isdecreased is paraesthesia, e.g. oral or skin.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the side effect isparaesthesia and the frequency is decreased by at least about 35%, inanother example by about 30-35%, in another example by about 25-30%, inanother example by about 20-25%, in another example by about 15-20%, inanother example by about 15-20%, in another example by 10-15%.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the side effect is dizzinessand the frequency is decreased by at least about 60%, in another exampleby about 55-60%, in another example by about 50-55%, in another exampleby about 45-50%, in another example by about 40-45%, in another exampleby about 35-40%, in another example by 30-35%, in another example byabout 25-30%, in another example by about 20-25%, in another example byabout 15-20%, in another example by about 10-15%, in another example byabout 5-10%.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the side effect is oralheadache and the frequency is decreased by at least about 75%, inanother example by about 70-75%, in another example by about 65-70%, inanother example by about 60-65%, in another example by about 55-60%, inanother example by about 50-55%, in another example by about 45-50%, inanother example by about 40-45%, in another example by about 35-40%, inanother example by 30-35%, in another example by about 25-30%, inanother example by about 20-25%, in another example by about 15-20%, inanother example by about 10-15%, in another example by about 5-10%.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the side effect ishypoaesthesia and the frequency is decreased by about 100%, in anotherexample by about 90-100%, in another example by about 80-90%, in anotherexample by about 70-80%, in another example by about 60-70%, in anotherexample by about 55-60%, in another example by about 50-55%, in anotherexample by about 45-50%, in another example by about 40-45%, in anotherexample by about 35-40%, in another example by 30-35%, in anotherexample by about 25-30%, in another example by about 20-25%, in anotherexample by about 15-20%, in another example by about 10-15%, in anotherexample by about 5-10%.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the side effect is facialhypoaesthesia and the frequency is decreased by about 100%, in anotherexample by about 90-100%, in another example by about 80-90%, in anotherexample by about 70-80%, in another example by about 60-70%, in anotherexample by about 55-60%, in another example by about 50-55%, in anotherexample by about 45-50%, in another example by about 40-45%, in anotherexample by about 35-40%, in another example by 30-35%, in anotherexample by about 25-30%, in another example by about 20-25%, in anotherexample by about 15-20%, in another example by about 10-15%, in anotherexample by about 5-10%.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the side effect is agastrointestinal disorder and is decreased by at least about 80% amount,in another example by about 70-80%, in another example by about 60-70%,in another example by about 55-60%, in another example by about 50-55%,in another example by about 45-50%, in another example by about 40-45%,in another example by about 35-40%, in another example by 30-35%, inanother example by about 25-30%, in another example by about 20-25%, inanother example by about 15-20%, in another example by about 10-15%, inanother example by about 5-10%.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the side effect is a nauseaand is decreased by at least about 65% amount, in another example byabout two thirds, in another example by about 60-65%, in another exampleby about 55-60%, in another example by about 50-55%, in another exampleby about 45-50%, in another example by about 40-45%, in another exampleby about 35-40%, in another example by 30-35%, in another example byabout 25-30%, in another example by about 20-25%, in another example byabout 15-20%, in another example by about 10-15%, in another example byabout 5-10%.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the side effect is abdominalpain and/or tenderness and the frequency is decreased by about 100%, inanother example by about 90-100%, in another example by about 80-90%, inanother example by about 70-80%, in another example by about 60-70%, inanother example by about 55-60%, in another example by about 50-55%, inanother example by about 45-50%, in another example by about 40-45%, inanother example by about 35-40%, in another example by 30-35%, inanother example by about 25-30%, in another example by about 20-25%, inanother example by about 15-20%, in another example by about 10-15%, inanother example by about 5-10%.

In some or any of embodiments, for example 2-4, 6, 7, 9-11, C-J, L-N,and Q, the human is a slow acetylator and the side effect is diarrheaand the frequency is decreased by about 100%, in another example byabout 90-100%, in another example by about 80-90%, in another example byabout 70-80%, in another example by about 60-70%, in another example byabout 55-60%, in another example by about 50-55%, in another example byabout 45-50%, in another example by about 40-45%, in another example byabout 35-40%, in another example by 30-35%, in another example by about25-30%, in another example by about 20-25%, in another example by about15-20%, in another example by about 10-15%, in another example by about5-10%.

In the following embodiments, the dose amount listed is the amount offree base in a dose whether the dose is administered as a free base oras a pharmaceutically acceptable salt thereof. In some or any ofembodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, and Q, the human isa slow acetylator and the optional pharmaceutically acceptable salt isphosphate. In some or any of embodiments, for example 2-4, 6, 7, 9-11,B-J, L-N, and Q, the human is a slow acetylator and the amount of3,4-DAP selected and/or administered is less than 40 mg total per day.In some or any of embodiments, for example 2-4, 6, 7, 9-11, B-J, L-N,and Q, the human is a slow acetylator and the amount of 3,4-DAP selectedand/or administered is less than 35 mg (base equivalent) total per day.In some or any of embodiments, for example 2-4, 6, 7, 9-11, B-J, L-N,and Q, the human is a slow acetylator and the amount of 3,4-DAP selectedand/or administered is less than 30 mg total per day. In some or any ofembodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, and Q, the human isa slow acetylator and the amount of 3,4-DAP selected and/or administeredis less than 25 mg total per day. In some or any of embodiments, forexample 2-4, 6, 7, 9-11, B-J, L-N, and Q, the human is a slow acetylatorand the amount of 3,4-DAP selected and/or administered is less than 20mg total per day. In some or any of embodiments, for example 2-4, 6, 7,9-11, B-J, L-N, and Q, the human is a slow acetylator and the amount of3,4-DAP selected and/or administered is less than 15 mg total per day.In some or any of embodiments, for example 2-4, 6, 7, 9-11, B-J, L-N,and Q, the human is a slow acetylator and the amount of 3,4-DAP selectedand/or administered is less than or equal to 12 mg total per day. Insome or any of embodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, andQ, the human is a slow acetylator and the amount of 3,4-DAP selectedand/or administered is less than or equal to 10 mg total per day. Insome or any of embodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, andQ, the human is a slow acetylator and the amount of 3,4-DAP selectedand/or administered is less than or equal to 9 mg total per day. In someor any of embodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, and Q, thehuman is a slow acetylator and the amount of 3,4-DAP selected and/oradministered is less than or equal to 6 mg total per day. In some or anyof embodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, and Q, the humanis a slow acetylator and the amount of 3,4-DAP selected and/oradministered is less than or equal to 5 mg total per day. In some or anyof embodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, and Q, the humanis a slow acetylator and the amount of 3,4-DAP selected and/oradministered is less than or equal to 3 mg total per day. In some or anyof embodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, and Q, the humanis a slow acetylator and the amount of 3,4-DAP selected and/oradministered is less than or equal to 2.5 mg total per day. In some orany of embodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, and Q, thehuman is a slow acetylator and the 3,4-DAP is selected and/oradministered one time a day. In some or any of embodiments, for example2-4, 6, 7, 9-11, B-J, L-N, and Q, the human is a slow acetylator and the3,4-DAP is selected and/or administered two times a day. In some or anyof embodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, and Q, the humanis a slow acetylator and the 3,4-DAP is selected and/or administeredthree times a day. In some or any of embodiments, for example 2-4, 6, 7,9-11, B-J, L-N, and Q, the human is a slow acetylator and the 3,4-DAP isselected and/or administered four times a day.

In the following embodiments, the dose amount listed is the amount offree base in a dose whether the dose is administered as a free base oras a pharmaceutically acceptable salt thereof. In some or any ofembodiments 2-4, 6, 7, 9-11, B-J, L-N, and Q, the optionalpharmaceutically acceptable salt is phosphate. In some or any ofembodiments 2-4, 6, 7, 9-11, B-J, L-N, and Q, the amount of 3,4-DAPselected and/or administered is less than about 40 mg total per day. Insome or any of embodiments 2-4, 6, 7, 9-11, B-J, L-N, and Q, the amountof 3,4-DAP selected and/or administered is less than about 35 mg totalper day. In some or any of embodiments 2-4, 6, 7, 9-11, B-J, L-N, and Q,the amount of 3,4-DAP selected and/or administered is less than about 30mg total per day. In some or any of embodiments 2-4, 6, 7, 9-11, B-J,L-N, and Q, the amount of 3,4-DAP selected and/or administered is lessthan about 25 mg total per day. In some or any of embodiments 2-4, 6, 7,9-11, B-J, L-N, and Q, the amount of 3,4-DAP selected and/oradministered is less than about 20 mg total per day. In some or any ofembodiments 2-4, 6, 7, 9-11, B-A, L-N, and Q, the amount of 3,4-DAPselected and/or administered is less than about 15 mg total per day. Insome or any of embodiments 2-4, 6, 7, 9-11, B-J, L-N, and Q, the amountof 3,4-DAP selected and/or administered is less than or equal to about12 mg total per day. In some or any of embodiments 2-4, 6, 7, 9-11, B-J,L-N, and Q, the amount of 3,4-DAP selected and/or administered is lessthan or equal to about 10 mg total per day. In some or any ofembodiments 2-4, 6, 7, 9-11, B-J, L-N, and Q, the amount of 3,4-DAPselected and/or administered is less than or equal to about 9 mg totalper day. In some or any of embodiments 2-4, 6, 7, 9-11, B-J, L-N, and Q,the amount of 3,4-DAP selected and/or administered is less than or equalto about 6 mg total per day. In some or any of embodiments 2-4, 6, 7,9-11, B-J, L-N, and Q, the amount of 3,4-DAP selected and/oradministered is less than or equal to about 5 mg total per day. In someor any of embodiments 2-4, 6, 7, 9-11, B-J, L-N, and Q, the amount of3,4-DAP selected and/or administered is less than or equal to about 3 mgtotal per day. In some or any of embodiments 2-4, 6, 7, 9-11, B-J, L-N,and Q, the amount of 3,4-DAP selected and/or administered is less thanor equal to about 2.5 mg total per day. In some or any of embodiments2-4, 6, 7, 9-11, B-J, L-N, and Q, the 3,4-DAP is selected and/oradministered one time a day. In some or any of embodiments 2-4, 6, 7,9-11, B-J, L-N, and Q, the 3,4-DAP is selected and/or administered twotimes a day. In some or any of embodiments 2-4, 6, 7, 9-11, B-J, L-N,and Q, the 3,4-DAP is selected and/or administered three times a day. Insome or any of embodiments 2-4, 6, 7, 9-11, B-J, L-N, and Q, the 3,4-DAPis selected and/or administered four times a day.

In the following embodiments, the dose amount listed is the amount offree base in a dose whether the dose is administered as a free base oras a pharmaceutically acceptable salt thereof. In some or any ofembodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, and Q, the optionalpharmaceutically acceptable salt is phosphate. In some or any ofembodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, and Q, the amount of3,4-DAP selected and/or administered is less than about 40 mg total perday, less than about 35 mg total per day, less than about 30 mg totalper day, less than about 25 mg total per day, less than about 20 mgtotal per day, less than about 15 mg total per day, less than about 12mg total per day, less than about 10 mg total per day, less than about 9mg total per day, less than about 6 mg total per day, less than about 5mg total per day, less than about 3 mg total per day, or less than about2.5 mg total per day. In some or any of embodiments, for example 2-4, 6,7, 9-11, B-J, L-N, and Q, the 3,4-DAP is selected and/or administeredone time a day. In some or any of embodiments, for example 2-4, 6, 7,9-11, B-J, L-N, and Q, the 3,4-DAP is selected and/or administered twotimes a day. In some or any of embodiments, for example 2-4, 6, 7, 9-11,B-J, L-N, and Q, the 3,4-DAP is selected and/or administered three timesa day. In some or any of embodiments, for example 2-4, 6, 7, 9-11, B-J,L-N, and Q, the 3,4-DAP is selected and/or administered four times aday.

In the following embodiments, the dose amount listed is the amount offree base in a dose whether the dose is administered as a free base oras a pharmaceutically acceptable salt thereof. In some or any ofembodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, and Q, the optionalpharmaceutically acceptable salt is phosphate. In some or any ofembodiments, for example 2-4, 6, 7, 9-11, B-J, L-N, and Q, the amount of3,4-DAP selected and/or administered is about 40 mg total per day, about35 mg total per day, about 30 mg total per day, about 25 mg total perday, about 20 mg total per day, about 15 mg total per day, about 12 mgtotal per day, about 10 mg total per day, about 9 mg total per day,about 6 mg total per day, about 5 mg total per day, about 3 mg total perday, or about 2.5 mg total per day. In some or any of embodiments, forexample 2-4, 6, 7, 9-11, B-J, L-N, and Q, the 3,4-DAP is selected and/oradministered one time a day. In some or any of embodiments, for example2-4, 6, 7, 9-11, B-J, L-N, and Q, the 3,4-DAP is selected and/oradministered two times a day. In some or any of embodiments, for example2-4, 6, 7, 9-11, B-J, L-N, and Q, the 3,4-DAP is selected and/oradministered three times a day. In some or any of embodiments, forexample 2-4, 6, 7, 9-11, B-J, L-N, and Q, the 3,4-DAP is selected and/oradministered four times a day.

Embodiments for a Fast Acetylator

In some or any of embodiments, for example 2-5, 7-11, C, D, and H-Q, thehuman is a fast acetylator and the 3,4-DAP is administered when thestomach is empty, for example, 60 min or more before or 120 min or moreafter ingestion of food.

In some or any of embodiments, for example 2-5, 7-11, C, D, and H-Q, thehuman is a fast acetylator and the 3,4-DAP is administered without food,for example, 60 min or more before or 120 min or more after ingestion offood.

In some or any of embodiments, for example 2-5, 7-11, C, D, and H-Q, thehuman is a fast acetylator and the 3,4-DAP is administered under fastingconditions, for example, 60 min or more before or 120 min or more afteringestion of food.

In some or any of embodiments, for example 2-5, 7-11, B, C, D, and H-Q,the human is a fast acetylator and the efficacy of 3,4-DAP is increasedwhen taken without food. In some or any of embodiments, for example 2-5,7-11, C, D, and H-Q, the human is a fast acetylator and the efficacy of3,4-DAP is increased by about 5% or more when taken without food. Inanother example the efficacy is increased by about 10% or more. Inanother example the efficacy is increased by about 15% or more. Inanother example the efficacy is increased by about 20% or more. Inanother example the efficacy is increased by about 25% or more. Inanother example the efficacy is increased by about 30% or more. Inanother example the efficacy is increased by about 35% or more. Inanother example the efficacy is increased by about 40% or more.

In the following embodiments, the dose amount listed is the amount offree base in a dose whether the dose is administered as a free base oras a pharmaceutically acceptable salt thereof. In some or any ofembodiments, for example 2-5, 7-11, B, C, D, and H-Q, the human is afast acetylator and the optional pharmaceutically acceptable salt isphosphate. In some embodiments, the human is a fast acetylator and theamount of 3,4-DAP selected and/or administered is greater than or equalto about 30 mg total per day. In some embodiments, the human is a fastacetylator and the amount of 3,4-DAP selected and/or administered isgreater than or equal to about 40 mg total per day. In some embodiments,the human is a fast acetylator and the amount of 3,4-DAP selected and/oradministered is greater than or equal to about 50 mg total per day. Insome embodiments, the human is a fast acetylator and the amount of3,4-DAP selected and/or administered is greater than or equal to about60 mg total per day. In some embodiments, the human is a fast acetylatorand the amount of 3,4-DAP selected and/or administered is greater thanor equal to about 80 mg total per day. In some embodiments, the human isa fast acetylator and the amount of 3,4-DAP selected and/or administeredis greater than or equal to about 100 mg total per day. In someembodiments, the human is a fast acetylator and the amount of 3,4-DAPselected and/or administered is greater than or equal to about 120 mgtotal per day. In some embodiments, the human is a fast acetylator andthe amount of 3,4-DAP selected and/or administered is greater than orequal to about 140 mg total per day. In some embodiments, the human is afast acetylator and the amount of 3,4-DAP selected and/or administeredis greater than or equal to about 160 mg total per day. In someembodiments, the human is a fast acetylator and the amount of 3,4-DAPselected and/or administered is greater than or equal to about 180 mgtotal per day. In some embodiments, the human is a fast acetylator andthe amount of 3,4-DAP selected and/or administered is greater than orequal to about 200 mg total per day. In some embodiments, the human is afast acetylator and the amount of 3,4-DAP selected and/or administeredis greater than or equal to about 220 mg total per day. In someembodiments, the human is a fast acetylator and the amount of 3,4-DAPselected and/or administered is greater than or equal to about 240 mgtotal per day. In some embodiments, the human is a fast acetylator andthe 3,4-DAP is administered one time a day. In some embodiments, thehuman is a fast acetylator and the 3,4-DAP is administered two times aday. In some embodiments, the human is a fast acetylator and the 3,4-DAPis administered three times a day. In some embodiments, the human is afast acetylator and the 3,4-DAP is administered four times a day. Insome embodiments, the human is a fast acetylator and the 3,4-DAP isadministered five times a day.

In the following embodiments, the dose amount listed is the amount offree base in a dose whether the dose is administered as a free base oras a pharmaceutically acceptable salt thereof. In some or anyembodiments 2-5, 7-11, B, C, D, and H-Q, the optional pharmaceuticallyacceptable salt is phosphate. In some or any of embodiments 2-5, 7-11,B, C, D, and H-Q, the amount of 3,4-DAP selected and/or administered isgreater than or equal to about 30 mg total per day. In some embodiments,the amount of 3,4-DAP selected and/or administered is greater than orequal to about 40 mg total per day. In some embodiments, the amount of3,4-DAP selected and/or administered is greater than or equal to about50 mg total per day. In some embodiments, the amount of 3,4-DAP selectedand/or administered is greater than or equal to about 60 mg total perday. In some embodiments, the amount of 3,4-DAP selected and/oradministered is greater than or equal to about 80 mg total per day. Insome embodiments, the amount of 3,4-DAP selected and or administered isgreater than or equal to about 100 mg total per day. In someembodiments, the amount of 3,4-DAP selected and/or administered isgreater than or equal to about 120 mg total per day. In someembodiments, the amount of 3,4-DAP selected and/or administered isgreater than or equal to about 140 mg total per day. In someembodiments, the amount of 3,4-DAP selected and/or administered isgreater than or equal to about 160 mg total per day. In someembodiments, the amount of 3,4-DAP selected and/or administered isgreater than or equal to about 180 mg total per day. In someembodiments, the amount of 3,4-DAP selected and/or administered isgreater than or equal to about 200 mg total per day. In someembodiments, the amount of 3,4-DAP selected and/or administered isgreater than or equal to about 220 mg total per day. In someembodiments, the amount of 3,4-DAP selected and/or administered isgreater than or equal to about 240 mg total per day. In someembodiments, the 3,4-DAP is selected and/or administered one time a day.In some embodiments, the 3,4-DAP is selected and/or administered twotimes a day. In some embodiments, the 3,4-DAP is selected and/oradministered three times a day. In some embodiments, the 3,4-DAP isselected and/or administered four times a day. In some embodiments, the3,4-DAP is administered five times a day.

In the following embodiments, the dose amount listed is the amount offree base in a dose whether the dose is administered as a free base oras a pharmaceutically acceptable salt thereof. In some or anyembodiments, for example 2-5, 7-11, B, C, D, and H-Q, the optionalpharmaceutically acceptable salt is phosphate. In some or any ofembodiments, for example 2-5, 7-11, B, C, D, and H-Q, the amount of3,4-DAP selected and/or administered is greater than or equal to about30 mg total per day, greater than or equal to about 40 mg total per day,greater than or equal to about 50 mg total per day, greater than orequal to about 60 mg total per day, greater than or equal to about 80 mgtotal per day, greater than or equal to about 100 mg total per day,greater than or equal to about 120 mg total per day, greater than orequal to about 140 mg total per day, greater than or equal to about 160mg total per day, greater than or equal to about 180 mg total per day,greater than or equal to about 200 mg per day, greater than or equal toabout 220 mg per day, or greater than or equal to about 240 mg per day.In some embodiments, the 3,4-DAP is selected and/or administered onetime a day. In some embodiments, the 3,4-DAP is selected and/oradministered two times a day. In some embodiments, the 3,4-DAP isselected and/or administered three times a day. In some embodiments, the3,4-DAP is selected and/or administered four times a day. In someembodiments, the 3,4-DAP is administered five times a day.

In the following embodiments, the dose amount listed is the amount offree base in a dose whether the dose is administered as a free base oras a pharmaceutically acceptable salt thereof. In some or anyembodiments, for example 2-5, 7-11, B, C, D, and H-Q, the optionalpharmaceutically acceptable salt is phosphate. In some or any ofembodiments, for example 2-5, 7-11, B, C, D, and H-Q, the amount of3,4-DAP selected and/or administered is about 30 mg total per day, about40 mg total per day, about 50 mg total per day, 60 mg total per day,about 80 mg total per day, about 100 mg total per day, about 120 mgtotal per day, about 140 mg total per day, about 160 mg total per day,about 180 mg total per day, about 200 mg per day, about 220 mg per day,or about 240 mg per day. In some embodiments, the 3,4-DAP is selectedand/or administered one time a day. In some embodiments, the 3,4-DAP isselected and/or administered two times a day. In some embodiments, the3,4-DAP is selected and/or administered three times a day. In someembodiments, the 3,4-DAP is selected and/or administered four times aday. In some embodiments, the 3,4-DAP is administered five times a day.

Embodiment 12

Disclosed herein is a method of treating a 3,4-DAP-sensitive disease ina subject, comprising administering 3,4-DAP or a pharmaceuticallyacceptable salt thereof; and determining the ratio of acetylated 3,4-DAPto 3,4-DAP in said subject using any method as described in any of theembodiments and examples herein, for example the 3,4-DAP test. In someor any embodiments, the method further comprises informing the subjectthat the amount of 3,4-DAP should be increased or decreased in order tooptimize efficacy or reduce the frequency and/or severity of sideeffects. In some or any embodiments, the subject has a ratio of about 15or less and is informed that 3,4-DAP should be taken with food. In someor any embodiments, the subject has a ratio of about 15 or less and isinformed that side effects will be reduced when 3,4-DAP is taken withfood. In some or any embodiments, the subject has a ratio of about 30 ormore and is informed that 3,4-DAP should be taken without food. In someor any embodiments, the subject has a ratio of about 30 or more and isinformed that efficacy will be increased when 3,4-DAP is taken withoutfood. In some or any embodiments, the method further comprisesincreasing or reducing the amount of 3,4-DAP, or pharmaceuticallyacceptable salt thereof, administered to the subject in order tooptimize efficacy or reduce the frequency and/or severity of sideeffects. In some or any embodiments, the acetylated 3,4-DAP isN-(4-aminopyridin-3-yl)acetamide. In some or any embodiments, for thesubject whose ratio is about 15 or less, 3,4-DAP is taken with food. Insome or any embodiments, for the subject whose ratio is about 30 ormore, 3,4-DAP is taken without food.

Embodiment 13

In some or any embodiments, the subject is a fast acetylator and isinformed that 3,4-diaminopyridine or a pharmaceutically acceptable saltthereof should be taken with food. In some or any embodiments, thesubject is a fast acetylator and 3,4-diaminopyridine or apharmaceutically acceptable salt thereof is administered with food.

Embodiment 14

Disclosed is a method comprising administering 3,4-diaminopyridine or apharmaceutically acceptable salt thereof, to a human in need thereof,and informing said human that the frequency and/or severity of sideeffect(s) of said 3,4-DAP or pharmaceutically acceptable salt thereofare decreased when it is ingested with food compared to when ingestedwithout food.

Embodiment 15

Disclosed is a method of dosing a subject with renal impairment. In someor any embodiments, the subject is started at a dose of about 10 mg freebase equivalent per day (regardless of whether free base or apharmaceutically acceptable salt is administered). In some or anyembodiments, 3,4-DAP, or a pharmaceutically acceptable salt thereof isadministered with food. In some or any embodiments, a subject with renalimpairment is treated as a slow metabolizer regardless of their NAT1and/or NAT2 phenotype and/or genotype.

In one example at least 99.5% pure 3,4-DAP is used. Any salt, includingthe phosphate and tartrate salts, and any crystalline form of 3,4-DAPmay be utilized according to the methods and compositions providedherein. A variety of salts are described in U.S. Patent Publication No.US20040106651, incorporated herein by reference in its entirety.

Disclosed is a method of determining whether a subject is a slow or fastacetylator of 3,4-DAP comprising determining the subject's NATpolymorphism phenotype or genotype. In another embodiment, the phenotypeand genotype can be determined using any of the embodiments and examplesas described herein.

Kits/Articles of Manufacture

For use in the therapeutic applications described herein, kits andarticles of manufacture are also described herein. In variousembodiments, such kits comprise a carrier, package, or container that iscompartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) comprising one of theseparate elements to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. In some embodiments, the containers are formed from a variety ofmaterials such as glass or plastic.

In some embodiments, the articles of manufacture provided herein containpackaging materials. Packaging materials for use in packagingpharmaceutical products include, but are not limited to, blister packs,bottles, tubes, inhalers, pumps, bags, vials, containers, syringes,bottles, and any packaging material suitable for a selected formulationand intended mode of administration and treatment.

In some embodiments, the container(s) described herein comprise one ormore compounds described herein, optionally in a composition or incombination with another agent as disclosed herein. The container(s)optionally have a sterile access port (for example in some embodimentsthe container is an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). Such kits optionallycomprise a compound with an identifying description or label orinstructions relating to its use in the methods described herein.

In some embodiments, a kit will comprises one or more additionalcontainers, each with one or more of various materials (such asreagents, optionally in concentrated form, and/or devices) desirablefrom a commercial and user standpoint for use of a compound describedherein. Non-limiting examples of such materials include, but are notlimited to, buffers, diluents, filters, needles, syringes; carrier,package, container, vial and/or tube labels listing contents and/orinstructions for use, and package inserts with instructions for use. Aset of instructions is optionally included.

In certain embodiments, a label is on or associated with the container.In some embodiments, a label is on a container when letters, numbers orother characters forming the label are attached, molded or etched intothe container itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In certain embodiments, a label indicatesthat the contents are to be used for a specific therapeutic application.In some embodiments, the label indicates directions for use of thecontents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. In some embodiments, the packcontains a metal or plastic foil, such as a blister pack. The pack ordispenser device is optionally accompanied by instructions foradministration. In some embodiments, the pack or dispenser isaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.In certain embodiments, such notice is, for example, the labelingapproved by the U.S. Food and Drug Administration for prescriptiondrugs, or the approved product insert. In some embodiments, compositionscontaining a compound provided herein are formulated in a compatiblepharmaceutical carrier and are placed in an appropriate containerlabeled for treatment of an indicated condition.

Terms

“About,” as used herein, unless otherwise indicated, refers to a valuethat is no more than 10% above or below the value being modified by theterm. For example, the term “about 1 hour” means a range of from 48 minto 72 min. Further, when the term “about” is used in relation to aspecified dosage amount or range, the term “about” indicates that thedosage amount or range specified is an approximate dosage amount orrange and that it includes not only the amount or range actuallyspecified, but those amounts or ranges that may also be safe andeffective amounts that are somewhat, e.g., 10%, outside the cited amountor range.

“Administration with food,” “administered under fed conditions,”“administered on a full stomach,” and like phrases, unless otherwiseindicated, means the drug is administered less than about one hourbefore food is ingested or less than about two hours after food isingested. In another example, these phrases mean that the drug isadministered less than about 30 min before food is ingested or less thanabout 45 min after food is ingested. In another example, these phrasesmean that the drug is administered less than about 15 min before food isingested or less than about 30 min after food is ingested. In anotherexample, the phrases have meanings as described in the embodimentsand/or examples herein.

“Administration without food,” “administered under fasting conditions,”“administered on an empty stomach,” and like phrases, unless otherwiseindicated, means the drug is administered more than one hour before foodis ingested or more than two hours after food is ingested. In anotherexample, these phrases mean that the drug is administered more than twohours before food is ingested or more than three hours after food isingested. In another example, the phrases have meanings as described inthe embodiments and/or examples herein.

As used herein, the term “bioavailability” refers to the fraction of anadministered dose of a drug entering systemic circulation. If the drugwere administered intravenously, then its bioavailability theoreticallywould be 100%. However, if the drug were administered via other routes(such as orally), then its bioavailability would be less than 100% as aresult of, for example, incomplete absorption in the GI tract,degradation or metabolism prior to absorption, and/or hepatic first passeffect.

“Fast alleles,” as used herein, for NAT2 mean alleles *4 (wildtype) andpotentially *13.

“Fast acetylator” or “fast metabolizer” is a person who has a plasmaconcentration of 0-5 ng/mL 3,4-DAP at 4 hours post dose. Alternatively,a fast acetylator is a person who has a ratio of greater than about 0.2,for example between about 0.2 and about 0.3, as calculated using thefollowing formula (AFMU+AAMU)/(AFMU+AAMU+1X+1U) after administration of150 mg caffeine. Alternatively, a fast acetylator is a person who has atleast one fast allele, in another embodiment, two fast NAT2 alleles.Alternatively, a fast metabolizer is as described in Table 4 or any ofthe embodiments or examples herein.

The term “high fat meal” refers generally to a meal of at least about700 kcal and at least about 45% fat (relative percentage of kcal whichare fat), or alternatively at least about 900 kcal and at least about50% fat. The term “high fat food” refers generally to a food comprisingat least 20 g of fat, or at least 25, 30, 35, 40, 45, or 50 g of fat,and/or at least about 45% or 50% fat. One FDA Guidance defines a“high-fat meal” as approximately 50% of total caloric content of themeal, whereas a “high-calorie meal” is approximately 800 to 1000calories. The FDA recommends a high-fat and high-calorie meal as a testmeal for food-effect bioavailability and fed bioequivalence studies.This test meal should derive approximately 150, 250, and 500-600calories from protein, carbohydrate and fat, respectively. An exampletest meal consists of two eggs fried in butter, two strips of bacon,four ounces of hash brown potatoes and eight ounces of whole milk.Substitution is possible if a similar amount of calories from protein,carbohydrate, and fat has comparable meal volume and viscosity (Guidancefor Industry, Food-Effect Bioavailability and Fed BioequivalenceStudies, U.S. Department of Health and Human Services, Food and DrugAdministration, Center for Drug Evaluation and Research (CDER), December2002).

The phrase “human whose phenotype is slow acetylation” and the phrase “asubject is a slow acetylator” are essentially equivalent in meaning.

The phrase “human whose phenotype is fast acetylation” and the phrase “asubject is a fast acetylator” are essentially equivalent in meaning.

“Intermediate acetylator” and “intermediate metabolizer” is a person whohas a plasma concentration of possibly 5-10 ng/mL at 4 hours post dose.Alternatively, an intermediate acetylator is a person who has one fastNAT2 allele and one slow allele. An intermediate acetylator is asubcategory of fast acetylator.

“Mean plasma concentration” means the average of readings ofconcentration in a series of plasma samples.

“NAT polymorphism phenotype” as used herein include slow, intermediate,and fast acetylation status.

“NAT polymorphism genotype” as used herein refers to the number of fastor slow alleles a person has. Two fast alleles mean the person is a fastacetylator. One fast and one slow mean the person is an intermediateacetylator which is a subcategory of fast acetylator. No fast allelesmean the person is a slow acetylator.

“Slow alleles,” as used herein, for NAT2 mean alleles *5, *6, and *7.

“Slow acetylator” or “slow metabolizer” is a person who has a plasmaconcentration of 10-26 ng/mL 3,4-DAP at 4 hours post dose.Alternatively, a slow acetylator is a person who has a ratio of about0.2 or less, for example between about 0.1 and about 0.2 (inclusive) ascalculated using the following formula (AFMU+AAMU)/(AFMU+AAMU+1X+1U)after administration of 150 mg caffeine. Alternatively, a slowacetylator is a person who has no fast NAT2 alleles. Alternatively, aslow metabolizer is as described in Table 4 or any of the embodiments orexamples herein.

For the purpose of defining the potential side effects of taking3,4-DAP, “nervous system disorders” include, but are not limited to,paraesthesia, paraesthesia oral, dizziness, hypoaesthesia oral,headache, dysgeusia, hypoaesthesia, and hypoaesthesia facial.

For the purpose of defining the potential side effects of taking3,4-DAP, “gastrointestinal disorders” include, but are not limited to,nausea, abdominal pain, upper abdominal pain, abdominal tenderness,constipation, diarrhea, and oropharyngeal pain.

For the purpose of defining the potential side effects of taking3,4-DAP, “general disorders and administration site conditions” include,but are not limited to, fatigue, catheter sire pain, and feeling hot.

For the purpose of defining the potential side effects of taking3,4-DAP, “infections and infestations” include, but are not limited to,gastroenteritis and nasopharyngitis.

For the purpose of defining the potential side effects of taking3,4-DAP, “skin and subcutaneous tissue disorders” include, but are notlimited to, acne and blister.

For the purpose of defining the potential side effects of taking3,4-DAP, “vascular disorders” include, but are not limited to, flushingand phlebitis.

For the purpose of defining the potential side effects of taking3,4-DAP, “cardiac disorders” include, but arc not limited to,presyncope.

For the purpose of defining the potential side effects of taking3,4-DAP, “musculoskeletal and connective tissue disorders” include, butare not limited to, musculoskeletal stiffness.

Any of the preceding methods may be carried out by providing oradministering 3,4-diaminopyridine in a container containing printedlabeling informing the patient of the change in absorption parametersdescribed above.

Optionally, the methods provided herein also comprise the step ofproviding to the patient in need thereof a therapeutically effectiveamount of 3,4-diaminopyridine. The therapeutically effective amount willvary depending on the condition to be treated, and can be readilydetermined by the treating physician based on improvement in desiredclinical symptoms.

ABBREVIATIONS

The abbreviations have the following meanings.

1U 1-methylurate 1X 1 -methylxanthine λ_(Z) Apparent terminalelimination rate constant 3,4-DAP 3,4-diaminopyridine; amifampridineAAMU 5-acetylamino-6-amino-3-methyluracil AE adverse event Ae Amount ofdrug excreted in urine AFMU 5-acetylamino-6-formylamino-3-methyluracilALT alanine aminotransferase API active pharmaceutical ingredient ASTaspartate aminotransferase ATP adenosine triphosphate AUC area under theplasma concentration-time curve AUC_(0-t) Area under the plasmaconcentration-time curve from time zero up to the last measurableconcentration AUC_(0-inf) Area under the plasma concentration-time curvefrom time zero to infinity % AUC_(extrap) Percentage of AUC that is dueto extrapolation from Tlast to infinity BMI body mass index BMN1253,4-diaminopyridine phosphate BUN blood urea nitrogen CO₂ carbon dioxideCHO Chinese hamster ovary CI confidence interval CL/F Apparent totalplasma clearance CL_(R) Renal clearance C_(max) Maximum observed plasmaconcentration CRF Case Report Form CRU Clinical Research Unit CVcoefficient of variation DMSO dimethylsulfoxide ECG electrocardiogramEGTA ethylene glycol tetraacetic acid Equiv equivalents FAM ™6-carboxy-fluorescine FBS fetal bovine sera fe Fraction of dose excretedin urine FSH follicle stimulating hormone GCP Good Clinical Practice GGTgamma-glutamyltransferase GI gastrointestinal or gastrointestine GLPGood Laboratory Practice HBsAg hepatitis B surface antigen hCG humanchorionic gonadotrophin HEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIV humanimmunodeficiency virus hKv human potassium channel ICH InternationalConference on Harmonization IP investigational product ITT intent totreat LC/MS/MS liquid chromatography with tandem mass spectrometricdetection LDH lactate dehydrogenase LEMS Lambert-Eaton myasthenicsyndrome LS least squares MedDRA Medical Dictionary for RegulatoryActivities min minutes mV milivolt NAT N-acetyl transferase PKpharmacokinetic QMG quantitative myasthenia gravis scorc QTc QT intervalcorrected QTcB QT interval with Bazett's correction RBC red blood cellSAE serious adverse event SD standard deviation SGOT serumglutamic-oxaloacetic transaminase SGPT serum glutamic-pyruvictransaminase t_(1/2) Apparent plasma terminal elimination half-life T3triiodothyronine T4 thyroxine TID three times a day T_(last) Time oflast quantifiable plasma concentration T_(max) Time of the maximumobserved plasma concentration TSH thyroid-stimulating hormone VIDvolunteer information document V_(z)/F Apparent volume of distributionat the terminal phase WBC white blood cell

Pharmaceutical Formulations

The formulations described herein are in one example administered asoral formulations. Oral formulations are in one example solidformulations such as capsules, tablets, pills and troches, or liquidformulations such as aqueous suspensions, elixirs and syrups. Thevarious form of 3,4-DAP described herein can be directly used as powder(micronized particles), granules, suspensions or solutions, or it may becombined together with other pharmaceutically acceptable ingredients inadmixing the components and optionally finely divide them, and thenfilling capsules, composed for example from hard or soft gelatin,compressing tablets, pills or troches, or suspend or dissolve them incarriers for suspensions, elixirs and syrups. Coatings may be appliedafter compression to form pills.

Pharmaceutically acceptable ingredients are well known for the varioustypes of formulation and may be, for example, binders such as natural orsynthetic polymers, excipients, lubricants, surfactants, sweetening andflavoring agents, coating materials, preservatives, dyes, thickeners,adjuvants, antimicrobial agents, antioxidants and carriers for thevarious formulation types. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that are approved by the U.S. Food and Drug Administrationor a corresponding foreign regulatory agency for administration tohumans. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the therapeutic compositions, its use intherapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The initial amount of 3,4-diaminopyridine used to prepare theformulation may be, for example, in the range of about 5 wt % to about15 wt % of the formulation, or in the range of about 7 wt % to about 13wt %, or in the ranges of about 7 wt % to about 9 wt %. Specific amountsof 3,4-DAP in a tablet formulation contemplated herein include 5 mg, 10mg, 15 mg, 20 mg, 25 mg, and 30 mg.

Binders assist in maintaining a solid formulation. In some cases,anhydrous binders are used to preserve the anhydrous state of polymorphforms. In some cases, the binder may act as a drying agent. Exemplarybinders include anhydrous dibasic calcium phosphate and its monohydrate.Other non-limiting examples of binders useful in a composition describedherein include gum tragacanth, acacia, starch, gelatine, and biologicaldegradable polymers such as homo- or co-polyesters of dicarboxylicacids, alkylene glycols, polyalkylene glycols and/or aliphatic hydroxylcarboxylic acids; homo- or co-polyamides of dicarboxylic acids, alkylenediamines, and/or aliphatic amino carboxylic acids; correspondingpolyester-polyamide-co-polymers, polyanhydrides, polyorthoesters,polyphosphazene and polycarbonates. The biological degradable polymersmay be linear, branched or cross-linked. Specific examples arepoly-glycolic acid, poly-lactic acid, and poly-d,l-lactide/glycolide.Other examples for polymers are water-soluble polymers such aspolyoxaalkylenes (polyoxaethylene, polyoxapropylene and mixed polymersthereof, poly-acrylamides and hydroxylalkylated polyacrylamides,poly-maleic acid and esters or -amides thereof, poly-acrylic acid andesters or -amides thereof, poly-vinylalcohol und esters or -ethersthereof, poly-vinylimidazole, poly-vinylpyrrolidon, und natural polymerslike chitosan.

Disintegration agents assist in rapid disintegration of solidformulations by absorbing water and expanding. Exemplary disintegrationagents include polyvinylpyrrolidone (PVP, e.g., sold under the namePOVIDONE), a cross-linked form of povidone (CPVP, e.g., sold under thename CROSPOVIDONE), a cross-linked form of sodium carboxymethylcellulose(NaCMC, e.g., sold under the name AC-DI-SOL), other modified celluloses,and modified starch. Tablets formulated with CPVP exhibited much morerapid disintegration than tablets formulated with PVP.

Lubricants improve stability, hardness and uniformity of solidformulations. Exemplary lubricants include stearyl fumarate andmagnesium stearate. Other non-limiting examples of lubricants includenatural or synthetic oils, fats, waxes, or fatty acid salts such asmagnesium stearate.

Optionally the stable formulations provided herein can also compriseother excipients such as mannitol, hydroxyl propyl cellulose,microcrystalline cellulose, or other non-reducing sugars such assucrose, trehalose, melezitose, planteose, and raffinose. Reducingsugars may react with 3,4-DAP. Other non-limiting examples of excipientsuseful in a composition described herein include phosphates such asdicalcium phosphate.

Surfactants for use in a composition described herein can be anionic,anionic, amphoteric or neutral. Nonlimiting examples of surfactantsuseful in a composition described herein include lecithin,phospholipids, octyl sulfate, decyl sulfate, dodecyl sulfate, tetradecylsulfate, hexadecyl sulfate and octadecyl sulfate, sodium oleate orsodium caprate, 1-acylaminoethane-2-sulfonic acids, such as1-octanoylaminoethane-2-sulfonic acid, 1-decanoylaminoethane-2-sulfonicacid, 1-dodecanoylaminoethane-2-sulfonic acid,1-tetradecanoylaminoethane-2-sulfonic acid,1-hexadecanoylaminoethane-2-sulfonic acid, and1-octadecanoylaminoethane-2-sulfonic acid, and taurocholic acid andtaurodeoxycholic acid, bile acids and their salts, such as cholic acid,deoxycholic acid and sodium glycocholates, sodium caprate or sodiumlaurate, sodium oleate, sodium lauryl sulphate, sodium cetyl sulphate,sulfated castor oil and sodium dioctylsulfosuccinate,cocamidopropylbetaine and laurylbetaine, fatty alcohols, cholesterols,glycerol mono- or distearate, glycerol mono- or dioleate and glycerolmono- or dipalmitate, and polyoxyethylene stearate.

Non-limiting examples of sweetening agents useful in a compositiondescribed herein include sucrose, fructose, lactose or aspartame.Non-limiting examples of flavoring agents for use in a compositiondescribed herein include peppermint, oil of wintergreen or fruit flavorssuch as cherry or orange flavor. Non-limiting examples of coatingmaterials for use in a composition described herein include gelatin,wax, shellac, sugar or other biological degradable polymers.Non-limiting examples of preservatives for use in a compositiondescribed herein include methyl or propylparabens, sorbic acid,chlorobutanol, phenol and thimerosal.

The 3,4-DAP used in a composition described herein can be formulated asthe free base or as a phosphate salt or alternatively as a tartratesalt; however, it is contemplated that other salt forms of 3,4-DAPpossess the desired biological activity, and consequently, other saltforms of 3,4-DAP can be used. Specifically, for example, 3,4-DAP saltscan be formed with inorganic or organic acids. Nonlimiting examples ofalternative 3,4-DAP salts forms includes 3,4-DAP salts of acetic acid,citric acid, oxalic acid, tartaric acid, fumaric acid, and mandelicacid.

Pharmaceutically acceptable base addition salts may be formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Pharmaceutically acceptable salts of compounds may also beprepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.Examples of metals used as cations are sodium, potassium, magnesium,ammonium, calcium, or ferric, and the like. Examples of suitable aminesinclude isopropylamine, trimethylamine, histidine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine.

Pharmaceutically acceptable acid addition salts include inorganic ororganic acid salts. Examples of suitable acid salts include thehydrochlorides, acetates, citrates, salicylates, nitrates, phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include, for example, acetic, citric, oxalic,tartaric, or mandelic acids, hydrochloric acid, hydrobromic acid,sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic,sulfo or phospho acids or N-substituted sulfamic acids, for exampleacetic acid, propionic acid, glycolic acid, succinic acid, maleic acid,hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid,tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid,glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid,2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinicacid; and with amino acids, such as the 20 alpha amino acids involved inthe synthesis of proteins in nature, for example glutamic acid oraspartic acid, and also with phenylacetic acid, methanesulfonic acid,ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane 1,2-disulfonicacid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene2-sulfonic acid, naphthalene 1,5 disulfonic acid, 2- or3-phosphoglycerate, glucose 6 phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid.

Exemplary stable oral formulations contain one or more of the followingadditional ingredients that improve the stability or othercharacteristics of the formulation: binder, disintegration agent, acidicantioxidant, or lubricant or combinations thereof. Exemplary stabletablet formulations include a binder and disintegration agent,optionally with an acidic antioxidant, and optionally further includinga lubricant. Exemplary concentrations of binder are between about 1 wt %to about 5 wt %, or between about 1.5 and 3 wt %; an exemplary weightratio of binder to 3,4-DAP is in the range of about 1:10 to about 1:20.Exemplary concentrations of disintegration agent are between about 1 wt% to about 20 wt %; an exemplary weight ratio of disintegration agent to3,4-DAP is in the range of about 1:5 to about 1:10. Exemplaryconcentrations of antioxidant are between about 1 wt % and about 3 wt %;an exemplary weight ratio of antioxidant to 3,4-DAP is in the range ofabout 1:5 to 1:30. In one example, ascorbic acid is the antioxidant andis used at a ratio to 3,4-DAP of less than 1:1, e.g. 1:2 or less, or1:10 or less. Exemplary concentrations of lubricant in a stable tabletformulation are between about 0.1 wt % and about 5 wt %; an exemplaryweight ratio of lubricant to 3,4-DAP is in the range of about 1:25 to1:65.

The stable formulations may be provided, e.g. as tablets or pills orcapsules in HDPE bottles provided with a desiccant capsule or pouch; orin foil-on-foil blister packaging, or in blister packaging comprisingsee-through polymer film, if commercially desirable.

Treatment of 3,4-DAP-Responsive Diseases Hyperphenylalaninemia,Neuropsychological or Neuropsychiatric Disorders

The methods provided herein can be used for treatment of 3,4-DAPresponsive conditions, including myasthenia gravis and myasthenicsyndromes (including Lambert-Eaton myasthenic syndrome, congenitalmyasthenia, and myasthenic syndromes of medicinal or toxic origin),improving the cognitive functions during aging, treatment of fatiguerelated to a neurological pathology, diseases affecting motor neuroncells, such as acute infectious poliomyelitis and its effects,Creutzfeldt-Jakob syndrome, some toxic and nutritional disorders, suchas those related to vitamin B deficiency, degeneration of motor neuronsas a result of exposure to certain compounds, such as aluminum, ordegenerative diseases, such as amyotrophic lateral sclerosis, primarylateral sclerosis, pre-senile dementia with attack on motor neurons,spinal muscular atrophies, olivoponto-cerebellar atrophy, Joseph'sdisease, Parkinson's disease, Huntington's chorea or Pick's disease.

The amount of 3,4-DAP needed varies considerably between individuals.Dosages of about 15 mg per day to about 60 mg per day have beendescribed.

In exemplary embodiments, it is contemplated that the methods providedherein will provide to a patient in need thereof, a daily dose ofbetween about 2.5 mg per day and 180 mg per day of 3,4-DAP. This dose isadjusted up or down depending on the efficacy being achieved by or theside effect(s) observed with the administration. The daily dose may beadministered in a single dose or alternatively may be administered inmultiple doses at conveniently spaced intervals. In exemplaryembodiments, the daily dose may be 2.5 mg per day, 3 mg per day, 5 mgper day, 7.5 mg per day, 9 mg per day, 10 mg per day, 12 mg per day,12.5 mg per day, 15 mg per day, 20 mg per day, 25 mg per day, 30 mg perday, 35 mg per day, 40 mg per day, 45 mg per day, 50 mg per day, 55 mgper day, 60 mg per day, 65 mg per day, 70 mg per day, 75 mg per day, 80mg per day, 85 mg per day, 90 mg per day, 95 mg per day, 100 mg per day,105 mg per day, 110 mg per day, 115 mg per day, 120 mg per day, 125 mgper day, 130 mg per day, 135 mg per day, 140 mg per day, 145 mg per day,150 mg per day, 155 mg per day, 160 mg per day, 165 mg per day, 170 mgper day, 175 mg per day, or 180 mg per day.

It is understood that the suitable dose of a 3,4-DAP will depend uponthe age, health and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired (i.e., the amount of decrease in side effects desired or theincrease in efficacy desired) in addition to a subject's fast or slowmetabolizer status. The frequency of dosing also is dependent onpharmacodynamic effects.

The frequency of 3,4-DAP dosing will depend on its pharmacokineticparameters and the routes of administration. The optimal pharmaceuticalformulation will be determined by one of skill in the art depending onthe route of administration and the desired dosage. See for exampleRemington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publ. Co,Easton Pa. 18042) pp 1435 1712, incorporated herein by reference. Suchformulations may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of the administered agents.Depending on the route of administration, a suitable dose may becalculated according to body weight, body surface areas or organ size.Further refinement of the calculations necessary to determine theappropriate treatment dose is routinely made by those of ordinary skillin the art without undue experimentation, especially in light of thedosage information and assays disclosed herein as well as thepharmacokinetic data observed in animals or human clinical trials.

The final dosage regimen will be determined by the attending physician,considering factors which modify the action of drugs, e.g., the drug'sspecific activity, severity of the damage and the responsiveness of thepatient, the age, condition, body weight, sex and diet of the patient,the severity of any infection, time of administration and other clinicalfactors, including the individual's status as a fast or slow acetylator.As studies are conducted, further information will emerge regardingappropriate dosage levels and duration of treatment for specificdiseases and conditions.

EXAMPLES

It should be appreciated by those of skill in the art that thetechniques disclosed in the examples which follow represent techniquesdiscovered by the inventor to function well in the practice of theinvention. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example A Bioavailability/Bioequivalence Study

Objectives

The primary objectives of the study were to evaluate the clinicaltolerance of a single 10 mg dose (equivalent base) oral intake of3,4-DAP salt in five healthy subjects and to determine the relativebioequivalence of a single 20 mg oral dose of 3,4-DAP administered as asalt or as a free base to 26 healthy subjects. The secondary objectiveswere to study pharmacokinetic parameters (elimination half andelimination constant), to compare the biological tolerance of both a 20mg oral intake of 3,4-DAP as a free base and as a salt, and to measureQTcf intervals at expected C_(max).

Overall Study Plan

Tolerance Evaluation:

This was an open and uncontrolled study of the clinical tolerance to alow dose of the phosphate salt. Subjects were hospitalized from 8 am to4 pm after fasting since midnight the previous night. At 8:30 am, one 10mg 3,4-DAP phosphate tablet was administered with a glass of water inthe presence of a supervising nurse. During their 8-hour stay, heartrate was continuously monitored, blood pressure was measured on eightoccasions (including baseline) and three urine samples were taken.

Bioequivalence Study:

This was a double-blind, randomized 2-way cross-over trial. The twoadministration periods were separated by a wash-out of at least 3 daysand not more than 10 days. At the beginning of each period, subjectswere hospitalized at 8 am (while fasting since midnight) for at least 12hours. At their arrival a urinary sample was taken and avenous catheterwas inserted into an antecubital vein. A first blood sample was drawn 15min before drug intake. At around 8:30 am drug was administered (as thephosphate salt or free base according to randomization) with a glass ofwater under the supervision of a nurse and subjects remained seated for15 min. Group A received two 10 mg tablets of 3,4-DAP phosphate. Group Breceived two 10 mg capsules of 3,4-DAP as a free base.

Two 5 mL blood samples were drawn at the following times after drugadministration: 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min,75 min, 90 min, 120 min, 240 min, 360 min, 480 min, and 660 min. At eachsampling time two 5 mL samples were drawn on Li²⁺ heparinized tubes.They were immediately centrifuged at room temperature. Serum was thenseparated and dispatched into three clearly identified aliquots whichwere frozen at −20° C. Two aliquots were used for drug assay and one waskept in the hospital ward.

Urine was collected at dosing and between 0-4 hours after dosing,between 4-8 hours after dosing, and between 8-11 hours after dosing.Subjects were then discharged from the hospital and instructed tocollect their urine at home from between 11-24 hours after dosing. Urinewas sampled in tubes without conservative. Total volume was noted andtwo 10 mL samples were frozen at −20 C (one used for dosages and onekept in the hospital).

12-lead ECGs (50 mm/s, 2 cm/mV) were recorded over 1 minute three timesprior to drug intake (HR-RR, QT and QTcf were averaged to determinebaseline values) and then at 15, 20, 30, 45, 60, 75, 90, 120, and 240min post dose. ECG parameters were recorded by using Cardionics™ ECG/VCGsoftware (version 3.3.0). Blood pressure was measured at 120 min postdosing.

Subjects remained fasting up to 4 hours post dosing when a standardlunch was provided. Adverse events were spontaneously notified andsubjects were discharged at around 8 pm with an appropriate device forurine sampling for between 11-24 hours post dosing. The following day,subjects were required to return to the hospital at 8 am for clinicalexamination, recording of any adverse event and final blood sampling at24 hours post dose.

An HPLC-electrochemical detection system was used to assay 3,4-DAP inserum samples. The scrum samples were prepared by solid phase extraction(SPE). The eluate from SPE was evaporated under nitrogen and the samplewas reconstituted with water acetonitrile (50/50 v/v). The extractedsample was analyzed by HPLC coupled with coulometric detection. TwoZorbax SB-CN Interchim columns (250 mm×4.6 mm, 5 μm), mounted in series,were used to chromatograph the sample. Results were specific andaccurate for 3,4-DAP with the linearity documented within 5.0-150 ng/mLconcentration range. The quantitation limit is 5.0 ng/mL.

Results

The following pharmacokinetic parameters were calculated from theindividual plasma concentration versus time profiles: AUC_(0-t),AUC_(0-inf), % AUC, C_(max), T_(max), t_(1/2), and λ_(Z).

3,4-DAP either administered as a phosphate salt or as a free base is ahighly variable drug with coefficient of variations largely exceeding30% for AUCs and C_(max). Half life was always less than 2.6 hours.C_(max) for 3,4-DAP as the phosphate salt ranged from 13 ng/mL to 167ng/mL demonstrating the variability of maximum plasma concentrationsbetween patients. See FIGS. 16-19 b for additional details of theresults.

Examples 1-3 Drug and Metabolite Concentrations in Rat, Dog, and HumanHepatocytes

Preparation of Cryopreserved and Fresh Hepatocytes

The objective of this hepatocyte study was to determine the extent ofmetabolism, complete metabolic profile and metabolite characterizationof amifampridine phosphate in several species with ¹⁴C-amifampridinephosphate for a cross species comparison. The following procedures wereused to study the metabolism in rat (Example 1), dog (Example 2), andhuman hepatocytes (Example 3).

The cryopreserved hepatocytes were removed from storage, quickly thawedin a 37° C. water bath for 1 to 2 min, and transferred to a 50 mL tubecontaining pre-warmed InVitroGRO HT medium (Celsis Inc., Baltimore). Thecell suspension was mixed by gentle inversion and the cells werepelleted by centrifugation at 50 to 75×g for 5 min. The supernatantmedium was removed and the cells were resuspended in Williams' Medium E.Hepatocyte viability was assessed by using trypan blue exclusion.Hepatocyte density was determined by counting cells with a hemocytometerand the density was adjusted with Williams' Medium E to achieve a finaldensity of 1,000,000 cells/mL. The fresh hepatocyte suspensions weremixed by gently inverting the container and centrifuged for 4 min topellet the cells (70 to 75×g for rat and dog hepatocytes; 85 to 90×g forhuman hepatocytes). The supernatant medium was removed and the cellswere resuspended in cold Williams' Medium E. The cell density was thendetermined using a hemocytometer and the density was adjusted withWilliams' Medium E to achieve a final density of 1,000,000 cells/mL.Several incubations with hepatocytes from dogs were also conducted at acell density of 2,000,000 cells/mL.

Fortification Solution Preparation

Fortification solutions for hepatocyte incubations were prepared byweighing and dissolving ¹⁴C-amifampridine and amifampridine in water togenerate 0.1, 1, and 5 mM stock solutions (100,000 dpm/mL). A 10 μLaddition of the appropriate fortification solution was used to generatefinal concentrations of 1, 10, and 50 μM in a 1 mL incubation volume.The radiolabel concentration was 100,000 dpm/mL in the initialincubations with hepatocytes from all species evaluated. In order toevaluate the possibility of low level metabolites, additionalincubations with higher concentrations of radioactivity were conductedwith rat, dog, and human hepatocytes prepared using 1 mM stock solutionsin water to achieve 10 μM and 1,000,000 dpm/mL in the incubationmixtures. Additional experimentation also included dog hepatocytes,incubated with 5 mM stock solutions in water to achieve 50 μM and1,000,000 dpm/mL in the final incubation mixtures.

Determination of the In Vitro Metabolism of ¹⁴C-Amifampridine fromPrimary Hepatocyte Suspensions

Primary hepatocytes (1×10⁶ hepatocytes/mL in Williams' Medium E) wereincubated with ¹⁴C-amifampridine (1, 10, or 50 μM and 100,000 dpm/mL) at37° C. in an atmosphere of 5% CO₂. Control incubations to monitor fornon-enzymatic degradation were conducted at all time points byincubating 10 μM ¹⁴C-amifampridine (100,000 dpm/mL) in the absence ofhepatocytes. To ascertain endogenous components present during LC-MSanalysis, control incubations were also conducted containing hepatocytesin Williams' Medium E without ¹⁴C-amifampridine present.

The hepatocyte suspension was gently swirled by hand and 1 mL wasdispensed into the appropriate well plates. An equal volume of Williams'Medium E was added to the wells for control incubations. The cells werepre-incubated for 15 min on a platform shaker in an incubator set tomaintain 37° C. in an atmosphere of 5% CO₂. Reactions were initiated byadding 10 μL of concentrated test article solution. Incubations wereterminated at 0, 30, 60, 120, and 240 min by adding 2 mL of acetonitrilecontaining 10% formic acid to the test article incubations (2:1dilution). For the time-0 incubations, the hepatocyte suspension sampleswere stopped by adding 2 mL of acetonitrile containing 1% formic acidprior to adding the test article. Samples were vortex mixed, and theprotein was removed by centrifugation at 1400×g for 10 min at 4° C. Thesupernatants (66% acetonitrile/33% media) were transferred to separatetubes and stored at approximately −20° C. until analysis.

In order to evaluate the possibility of low level metabolites,additional incubations were conducted with higher concentrations ofradioactivity. ¹⁴C-amifampridine (10 μM and 1,000,000 dpm/mL) wasincubated with rat, dog, and human hepatocytes (1×10⁶ hepatocytes/mL)for 240 min following the procedures above. Dog hepatocytes were alsoincubated with 50 μM ¹⁴C-amifampridine (1,000,000 dpm/mL) and 2×10⁶hepatocytes/mL for 240 min.

Preparation of Samples for Radio-HPLC Analysis

The supernatant samples initially were directly analyzed by theradio-HPLC method measured below:

HPLC System: Shimadzu System UV wavelength: 254 nm Radioactivitydetector: Packard 500 Series Cell type: Time resolved-liquidscintillation counting (TR-LSC ™) Cell volume: 0.5 mL Scintillationfluid: Ultima Flo M Scintillation fluid flow rate: 3 mL/minute Columns:Waters Atlantis HILIC Silica, 4.6 × 150 mm, 5 μm Guard column: WatersAtlantis HILIC Silica, 4.6 × 20 mm, 5 μm Column temperature: 30° C.Injection volume: 3, 4, or 15 μt Mobile Phase A: 10 mM ammonium formate,pH 3 Mobile Phase B: acetonitrile Flow rate: 1 mL/minute Gradient: Time(mins) Mobile Phase (% A:% B)  0  5:95  2  5:95 20 50:50 25 50:50 26 5:95 34  5:95Quantitation of Test Article and its Metabolites by Radio-HPLC Analysis

Samples from hepatocyte incubations were analyzed for metabolites of¹⁴C-amifampridine by radio-HPLC. In the incubations containing 100,000dpm/mL, due to low analyte peak area to background ratios, all the peakswith an area>5% of total radioactivity in the chromatogram wereintegrated and were considered to constitute a region of interest (ROI).In the incubations containing 1,000,000 dpm/mL, all the peaks with anarea>1% of total radioactivity in the chromatogram were integrated andwere considered to constitute the ROI.

EXAMPLE 1. Drug and Metabolite Concentration in Rat Hepatocytes: Resultsare described in FIG. 1. M1 is N-(4-aminopyridin-3-yl)acetamide.

EXAMPLE 2. Drug Concentration in Dog Hepatocytes: Results are describedin FIG. 2.

EXAMPLE 3. Drug and Metabolite Concentration in Human Hepatocytes:Results are described in FIG. 3. M1 is N-(4-aminopyridin-3-yl)acetamide.

Example 4 N-(4-Aminopyridin-3-yl)acetamide Metabolite ProductionIn-Vivo: Rat PK Study

The objective of this study was to determine the absolutebioavailability of amifampridine phosphate and the pharmacokinetics ofamifampridine and its 3-N-acetyl metabolite upon single-dose IV andsingle-day oral dose (TID) administration.

Amifampridine phosphate was administered by IV bolus at 0.8 mg/kg (insaline) to 6 rats/sex and by oral gavage (in water) TID (approximately 6hours apart) at dose levels of 2, 8, and 25 mg/kg/dose (equivalent to 6,24, and 75 mg/kg/day) to 12 rats/sex/group, with oral Dose 1 given tofasted rats and Dose 2 and 3 with food ad libitum. For IV bolus dose andoral Dose 1 (dose administration No. 1), blood samples were collectedfrom three animals/sex/timepoint at predose and 0.083 (IV only), 0.17,0.25, 0.33, 0.5, 0.75, 1, 1.25 (IV only), 1.5, 2, 2.5, 3, and 6 hourspost-dose; 0.17, 0.5, and 6 hours following the second oral dose (doseadministration No. 2); and 0.17, 0.25, 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 4,6, 8, and 10 hours following the third oral dose (dose administrationNo. 3). The second and third oral doses were given at 6 and 12 hours,respectively, after the first dose. LC-MS/MS was used to quantifyamifampridine, 3-N-acetyl and 4-N-acetyl amifampridine metabolites inrat plasma samples. Due to low concentrations of the 4-N-acetylmetabolite (Cmax<25 ng/mL) detected in plasma, PK analysis was notperformed for this metabolite. Noncompartmental analysis was applied tothe mean plasma amifampridine and 3-N-acetyl metabolite concentrationdata for male and female rats.

Results are described in FIGS. 4 and 5. FIG. 4 depicts the concentrationtime curve of 3,4-DAP and its metabolite at various doses. FIG. 5 showsthe C_(max) and AUC ratios of metabolite to 3,4-DAP phosphate afterfirst dose in a fasted state.

Example 5 In Vitro Inhibitory Activity of 3,4-DAP Phosphate andN-(4-aminopyridin-3-yl) acetamide HCl in CHO Cells TransientlyTransfected with hKv1.7

Chemicals used in solution preparation were purchased from Sigma-Aldrich(St. Louis, Mo.), unless otherwise noted, and were of ACS reagent gradepurity or higher. Stock solutions of each test article (100.104 mM and1.00 mM 3,4-DAP phosphate and 100.023 mM and 336.335 mMN-(4-aminopyridin-3-yl) acetamide HCl) were prepared in sterile water,stored refrigerated and used within one day. Test article concentrationswere prepared fresh daily by diluting stock solutions intoHEPES-buffered physiological saline (HB-PS): 137 mM NaCl; 4.0 mM KCl;1.8 mM CaCl₂; 1 mM MgCl₂; 10 mM HEPES; 10 mM Glucose; and adjusted to pH7.4 with NaOH (prepared weekly and refrigerated). All test articlesolutions contained 3% sterile water. The test articles were prepared insufficient volume to run the assay (typically 50 mL).

CHO cells, transiently transfected with hKv1.7, were cryopreserved in90% FBS and 10% DMSO. Frozen vials of cells were thawed rapidly in a 37°C. water bath. Cells were transferred to a 15 mL conical tube, 10 mL ofgrowth media (Ham's/F12-PS/10% FBS) was added, the suspension was gentlymixed and the tube was centrifuged at approximately 250×g for 5 min. Themedium was removed, the cell pellet was resuspended in 20 mL of medium,and cells were triturated to disperse cell clumps. For electrophysiologyuse, 2 mL of the cell suspension was plated in each 35 mm cell culturedish. Recording began 2-3 hours after plating once the cells settled outof the media and adhered to the bottom of the culture dish. Forelectrophysiological recording, the cell culture media was removed andreplaced with continuously flowing vehicle solution.

Cells were transferred to the recording chamber and superfused withvehicle control solution. Pipette solution for whole cell recordings,designed to mimic the intracellular conditions of the CHO cells, was 130mM potassium aspartate, 5 mM MgCl₂, 5 mM EGTA, 4 mM ATP and 10 mM HEPESand adjusted to pH 7.2 with KOH. Pipette solution was prepared inbatches, aliquoted, and stored frozen; a fresh aliquot was thawed eachday. The recording chamber and vehicle solution were maintained at roomtemperature. Patch pipettes were made from glass capillary tubing usinga P-97 micropipette puller (Sutter Instruments, CA) and were filled withpipette solution. A commercial patch clamp amplifier was used for wholecell recordings. Before digitization, current records were low-passfiltered at one-fifth of the sampling frequency. One or two test articleconcentrations were applied sequentially (without washout between testsubstance concentrations) in ascending order, to each cell (n≥2 wheren=number of observations). Onset and steady state inhibition of hKv1.7current due to the test article was measured using a pulse pattern withfixed amplitudes (depolarization: +10 mV amplitude, 300 ms duration)repeated at 10-second intervals from a holding potential of −80 mV.Current amplitude was measured at the end of the step to +10 mV. Currentwas monitored until a new steady state was achieved. A steady state wasmaintained for at least 30 seconds before applying the test article. For3,4-DAP phosphate, concentration-response data were fit to an equationof the form: % Inhibition={1−1/[1+([Test]/IC₅₀)^(N)]}*100 where [Test]is the test article concentration, IC₅₀ is the test articleconcentration at half-maximal inhibition, N is the Hill coefficient, and% Inhibition is the percentage of current inhibited at each test articleconcentration. Nonlinear least squares fits were solved with the Solveradd-in for Excel 2000, or later (Microsoft, WA). The IC₅₀ for sustainedcurrent was calculated for 3,4-DAP phosphate salt.

For 3,4-DAP phosphate salt, the concentration-response relationship wasevaluated at seven concentrations (1, 10, 30, 100, 300, 1000 and 3000μM) and for N-(4-aminopyridin-3-yl) acetamide HCl salt, theconcentration-response relationship was evaluated at threeconcentrations (100, 1000 and 3000 μM). For N-(4-aminopyridin-3-yl)acetamide HCl salt, testing was also conducted at 10,000 μM (n=2);however, visible precipitate was observed in this formulation andtherefore the data was not useable. The concentration-response data arepresented in FIG. 20.

Example 6 In Vitro Inhibitory Activity of 3,4-DAP Phosphate andN-(4-aminopyridin-3-yl) acetamide HCl in CHO or HEK293 Cells TransientlyTransfected with hKv1.1, hKv1.2, hKv1.3, hKv1.4, hKv1.1, or hKv1.5

The objective of this study was to examine the in vitro effects anddetermine the IC₅₀ of 3,4-DAP phosphate and the metaboliteN-(4-aminopyridin-3-yl) acetamide HCl on the following ion channels:cloned human Kv1.1 potassium channel (encoded by the human KCNA1 gene,expressed in HEK293 cells), cloned human Kv1.2 potassium channel(encoded by the human KCNA2 gene, expressed in HEK293 cells), clonedhuman Kv1.3 potassium channel (encoded by the human KCNA3 gene,expressed in CHO cells), cloned human Kv1.4 potassium channel (encodedby the human KCNA4 gene, expressed in HEK293 cells), cloned human Kv1.5potassium channel (encoded by the human KCNA5 gene and expressed in CHOcells), responsible for I_(Kur), ultra-rapid delayed rectifier potassiumcurrent.

Chemicals used in solution preparation were purchased from Sigma-Aldrich(St. Louis, Mo.), unless otherwise noted, and were of ACS reagent gradepurity or higher. Stock solutions of each test article (100 mM, 30 mMand 1.00 mM 3,4-DAP phosphate and 100 mMN-(4-aminopyridin-3-yl)acetamide) were prepared in sterile water andstored refrigerated. Test article concentrations were prepared freshdaily by diluting stock solutions into HEPES-buffered physiologicalsaline (HB-PS): 137 mM NaCl; 4.0 mM KCl; 1.8 mM CaCl₂; 1 mM MgCl₂; 10 mMHEPES; 10 mM Glucose; and adjusted to pH 7.4 with NaOH (prepared weeklyand refrigerated). All test article solutions contained 3% sterilewater. The test articles were prepared in sufficient volume to run theassay (typically 10 mL)

HEK293 cells were stably transfected with the appropriate ion channelcDNA encoding the pore-forming channel subunit. Stable transfectantswere selected using the G418-resistance gene incorporated into theexpression plasmid. Selection pressure was maintained with G418 in theculture medium. Cells were cultured in Dulbecco's Modified EagleMedium/Nutrient Mixture F-12 (D-MEM/F-12) supplemented with 10% fetalbovine serum, 100 U/mL penicillin G sodium, 100 μg/mL streptomycinsulfate and 500 μg/mL G418. CHO cells were stably transfected with theappropriate ion channel cDNA(s). Cells were cultured in Ham's F-12supplemented with 10% fetal bovine serum, 100 U/mL penicillin G sodium,100 μg/mL streptomycin sulfate, and the appropriate selectionantibiotics.

Before testing, cells in culture dishes were treated with trypsin. Cellswere transferred to a 15-mL conical tube and the tube was centrifuged atapproximately 1,300 rpm for 1.5 min. The medium was removed, the cellpellet was resuspended in 10 mL DMEM/F12, and cells were triturated todisperse cell clumps. Cells in suspension were allowed to recover for 10min in a tissue culture incubator set at 37° C. in a humidified 95% air,5% CO₂ atmosphere. The cells were then centrifuged for 1.5 min at 1,300rpm. The supernatant was removed, replaced with 10 mL HB-PS, and thecells were gently resuspended. The tube of cells was inverted once ortwice before once again being centrifuged for 1.5 min at 1,300 rpm. Thesupernatant was removed and the pellet was resuspended in ˜120 μL HB-PSand transferred to a 1.5 mL Eppendorf tube, which was then loaded ontothe PatchXpress machine. The target cell concentration was approximately8.3 million cells/mL.

Cells were transferred to the recording well and superfused with vehiclecontrol solution. Pipette solution for whole cell recordings, designedto mimic the intracellular conditions of the cells, was 130 mM potassiumaspartate, 5 mM MgCl₂, 5 mM EGTA, 4 mM ATP and 10 mM HEPES and adjustedto pH 7.2 with KOH. Pipette solution was prepared in batches, aliquoted,and stored frozen; a fresh aliquot was thawed each day. The recordingchamber and vehicle solution were maintained at room temperature.

In preparation for a recording session, intracellular solution wasloaded into the intracellular compartments of the Sealchip₁₆. Cellsuspension was pipetted into the extracellular compartments of theSealchip₁₆. After establishment of a whole-cell configuration, membranecurrents were recorded using dual-channel patch clamp amplifiers in thePatchXpress®. Before digitization, the current records were low-passfiltered at one-fifth of the sampling frequency.

One or two test article concentrations were applied sequentially(without washout between test substance concentrations) in ascendingorder, to each cell (n≥2 where n number of observations). Onset andsteady state inhibition of current due to the test article was measuredusing a pulse pattern with fixed amplitudes (indicated below) repeatedat 10-second intervals from a holding potential of −80 mV. A steadystate was maintained for at least 30 seconds before applying the testarticle. Current was monitored throughout the experiment.

Location of current Channel Voltage Step Duration amplitude measurementKv1.1 +40 mV 300 ms Peak current Kv1.2 +40 mV 300 ms Peak current Kv1.3+20 mV 300 ms End of voltage step Kv1.4 +20 mV 200 ms Peak current Kv1.5+20 mV 300 ms End of voltage step

For 3,4-DAP phosphate, concentration-response data were fit to anequation of the form: % Inhibition={1−1/[1+([Test]/IC₅₀)^(N)]}*100 where[Test] is the test article concentration, IC₅₀ is the test articleconcentration at half-maximal inhibition, N is the Hill coefficient, and% Inhibition is the percentage of current inhibited at each test articleconcentration. Nonlinear least squares fits were solved with the Solveradd-in for Excel 2000, or later (Microsoft, WA). The IC₅₀ for sustainedcurrent was calculated for 3,4-DAP phosphate salt.

For 3,4-DAP phosphate, the concentration-response relationship wasevaluated at seven concentrations (1, 10, 30, 100, 300, 1000 and 3000μM) and for N-(4-aminopyridin-3-yl) acetamide HCl, theconcentration-response relationship was evaluated at threeconcentrations (100, 1000 and 3000 μM). The concentration-response dataare presented in FIGS. 21a and b.

Example 7 Inhibition of NAT Enzymes with Acetaminophen

Fast acetylators of 3,4-diaminopyridine are expected to have reducedefficacy when compared to slow acetylators of 3,4-diaminopyridine since3,4-diaminopyridine is metabolized more quickly in fast acetylators. Itis therefore useful to inhibit NAT2 in order to increase drug levels infast acetylators. (See Pharmacogenetics 1998, 8(6), 553-9.)

Example 10 Randomized, Open-Label, Two-Treatment, Two-Period CrossoverStudy to Evaluate the Effect of Food on Relative Bioavailability ofAmifampridine Phosphate (3,4-Diaminopyridine Phosphate) in HealthySubjects

Objectives

The primary objectives of the study were to compare the effect of foodon the relative bioavailability of a single dose of amifampridinephosphate, 2 tablets administered orally, during fasting and fedconditions in healthy subjects. The secondary objective of the study wasto assess the safety and tolerability of a single dose of amifampridinephosphate, 2 tablets administered orally, during fasting and fedconditions in healthy subjects.

Overall Study Plan

This was a randomized, open-label, 2-treatment, 2-period crossover studywith 44 completed subjects to assess the safety, tolerability, and theeffect of food on the relative bioavailability of a single dose ofamifampridine phosphate. Each subject received a total of 2 single dosesof amifampridine phosphate as follows:

-   -   Treatment A: a single dose of amifampridine phosphate consisting        of 2 tablets (20 mg active pharmaceutical ingredient; API)        administered orally in a fasting state (overnight fast of at        least 10 hours)    -   Treatment B: a single dose of amifampridine phosphate consisting        of 2 tablets (20 mg API) administered orally in a fed state        (overnight fast of at least 10 hours, followed by a high fat        breakfast) 30 min after start of the meal        Subjects were randomized 1:1 to receive the 2 doses as follows:

Treatment Group 1: Treatment A then B

Treatment Group 2: Treatment B then A

The study design is presented in the figure below. Potential subjectswere screened up to 27 days prior to Check-in for the first study dose.For the Treatment Period 1, subjects resided in the CRU from theafternoon of Day −1 through the morning of Day 2 (approximately 24 hourspost dose). Subjects were treated on Day 1. For the Treatment Period 2,subjects were asked to return on the afternoon of Day 7 for Check-in andremain at the CRU through the morning of Day 9 (approximately 24 hourspost dose). Subjects were treated on Day 8. A Washout Period of 6 (up to10) days separated each dose administration, beginning on Day 2. A poststudy assessment was performed 5 to 7 days after the last dose.

Discussion of Study Design

The randomized, 2-period, 2-sequence crossover design used for the studyallowed the dietary status effect to be distinguished from othereffects. The study design is well accepted in the assessment of foodeffects on drug bioavailability and safety. This study design allowedfor healthy subjects to cross-over to alternate fed/fasted statesallowing for a direct comparison of outcomes.

Selection of Subject Population

Healthy adult volunteers were selected for participation in this studyincluding male or female subjects between the ages of 18 and 65inclusive, with a body mass index (BMI) between 18.5 and 30 kg/m²inclusive. Good health was evidenced by physical examination, clinicallaboratory evaluations (hematology, chemistry, and urinalysis), andelectrocardiogram (ECG). Individuals who met the following exclusioncriteria were not eligible to participate in this study: subjects usingmedication that prolongs the QT/QTc interval; subjects who had priorexposure to amifampridine (base or phosphate); subjects with a historypalpitations, epilepsy, seizures, unexplained syncopal episodes,arrhythmias, or risk factors for torsade de pointes; subjects who had,or had a history of, any clinically significant neurological,gastrointestinal, renal, hepatic, cardiovascular, psychiatric,respiratory, metabolic, endocrine, hematological, or other majordisorders as determined by the investigator; an ECG at Screening thatshowed any of the following: sinus arrhythmia with unacceptable ratevariation (e.g., >20% RR variability), excessive heart rate variation atrest, QTcB interval>450 ns confirmed by a repeat ECG, PR interval>210ms, QRS interval>120 ms at or under age 35 years or >110 ms over age 35years, early repolarization pattern that increased the risk ofparticipating in the study, or an abnormality in the 12-lead ECG atScreening that increased the risk of participating in the study.

Treatments and Administration

Treatments Administered

Amifampridine phosphate was provided in tablets containing theequivalent of 10 mg of amifampridine free base. In each treatmentperiod, subjects received a single dose consisting of 2 intact tabletsof amifampridine phosphate (equivalent of 10 mg of amifampridine each)administered orally with 240 mL of water. Each tablet containedamifampridine phosphate, microcrystalline cellulose, colloidal anhydroussilica, and calcium stearate. There was no comparator product for thisstudy.

For Treatment A, subjects were administered the dose with 240 mL ofwater, following an overnight fast of at least 10 hours. No food wasallowed for at least 4 hours post dose and no water was allowed for atleast 1 hour pre or post dose.

For Treatment B, following an overnight fast of at least 10 hours,subjects consumed a high-fat (approximately 60% of total caloric contentof the meal) and high-calorie (800 to 1,000 calories) meal. The mealderived 150 calories from protein, 250 calories from carbohydrate, and500 to 600 calories from fat. The meal was provided 30 min beforedosing, caten at a steady rate, and was completed approximately 10 minprior to dosing. The IP was administered 30 min after start of the mealand was administered with 240 mL of water. No food was allowed for atleast 4 hours post dose.

Subjects received standardized meals scheduled at approximately the sametime in each treatment period and water was allowed as desired exceptfor one hour before and after drug administration. Subjects receivedtreatments while in a standing position and were not allowed to liesupine for 2 hours post dose, except for study assessments or ifclinically indicated.

Method of Assigning Subjects to Treatment Groups

On Day −1, subjects were randomized to receive 2 single doses ofamifampridine phosphate in Treatment Group 1 or Treatment Group 2.Treatment Group 1 received Treatment A then B, while Treatment Group 2received Treatment B then A.

Treatment A: 2 intact tablets administered orally to subjects in afasting state.

Treatment B: 2 intact tablets administered orally to subjects in a fedstate.

Selection of Doses in the Study

Standard guidelines for food effect bioavailability studies recommendedutilizing the highest strength of a drug product intended to bemarketed, unless safety concerns warranted use of a lower strengthdosage form. Amifampridine phosphate received marketing approval by theEuropean Commission for the symptomatic treatment of patients with LEMSat a maximum single dose of up to 20 mg amifampridine. Safety of a 20 mgdose of amifampridine phosphate is based on nonclinical data andprevious human experience in healthy subjects and patients withneurological disorders.

Fasting Conditions

For treatment A, subjects receiving treatments administered underfasting conditions were dosed after they completed a minimum 10-hourovernight fast. The subjects continued to fast for 4 hours post dose.Water was allowed ad lib during the study except for 1 hour priorthrough 1 hour post-dose.

Subjects received standardized meals scheduled at approximately the sametime in each treatment period and water was allowed as desired exceptfor one hour before and after drug administration. Subjects receivedtreatments while in a standing position and were not allowed to liesupine for 2 hours post dose, except for study assessments or ifclinically indicated.

Non-Fasting Conditions

For Treatment B, following an overnight fast of at least 10 hours,subjects consumed a high-fat (approximately 60% of total caloric contentof the meal) and high-calorie (800 to 1,000 calories) meal. The mealderived 150 calories from protein, 250 calories from carbohydrate, and500 to 600 calories from fat. The meal was provided 30 min beforedosing, eaten at a steady rate, and was completed approximately 10 minprior to dosing. The TP was administered 30 min after start of the mealand was administered with 240 mL of water. No food was allowed for atleast 4 hours post dose.

Subjects received standardized meals scheduled at approximately the sametime in each treatment period and water was allowed as desired exceptfor one hour before and after drug administration. Subjects receivedtreatments while in a standing position and were not allowed to liesupine for 2 hours post dose, except for study assessments or ifclinically indicated.

Duration of Treatment

For the Treatment Period 1, all subjects received a single dose ofamifampridine phosphate on Day 1. A Washout Period of 6 days separatedeach dose administration, beginning on Day 2. For Treatment Period 2,all subjects received a single dose of amifampridine phosphate on Day 8.

Efficacy and Safety Variables

Safety Measurements Assessed

Safety was evaluated for all subjects who take at least one dose of3,4-DAP. The schedule for these assessments is shown in FIG. 15. Safetywas evaluated by recording the incidence of ad dverse events (AEs),physical examination, vital signs, concomitant medications, clinicallaboratory assessments, and 12-lead electrocardiogram (ECG).

Pharmacokinetic Measures

Blood samples (approximately 1×6 mL) were taken by venipuncture orcannulation of a forearm vein(s). The samples were collected intolithium heparin Vacutainer™ tubes and, after mixing, were placed in acool box containing crushed ice/water. Blood samples were centrifuged,within 1 hour of collection, at 1500 g for 10 min at approximately 4° C.For each sample, 1 mL of the separated plasma was transferred into eachof two, 5 mL polypropylene tubes, maintained at 0 to 4° C. prior tobeing stored (within 2 hours of collection) and subsequently stored atapproximately −70° C. until quantification by liquid chromatographytandem mass spectrometric (LC-MS/MS) analysis. Collection times andwindows for each sample collection time are listed in the followingtable. Each draw was to be completed, even if collected outside of agiven window.

Plasma Pharmacokinetic Assessment Times and Sampling Windows CollectionTime Window 90 min prior to dose ±5 Min 10, 15, 30, 45, 60, 75 min, 1.5,2, 4, and 6 hours post dose ±5 Min 8, 10, and 12 hours post dose ±10 Min18 and 24 hours post dose ±15 Min

Urine samples (≥approximately 40 mL) were collected at the followingtimes and intervals: ≤90 min prior to dose (single sample), 0 to 4hours, 4 to 8 hours, and 8 to 24 hours post dose to quantify total doseexcreted of un-metabolized amifampridine and the major metabolite3-N-acetyl amifampridine. During each collection period, the containerswere stored in a refrigerator at 2 to 8° C. The weight (g) of each urinecollection was recorded prior to removal of 2 sub-samples (20 to 25 mL)into suitably labelled polypropylene containers, which were storedwithin 4 hours of the end of the collection, at approximately −70° C.until quantification by LC-MS/MS. The remaining urine was discarded.

The PK parameters determined for Amifampridine and 3-N-AcetylAmifampridine were: AUC_(0-t), AUC_(0-inf), % AUC_(extrap), C_(max),T_(max), t_(1/2), T_(last), λ_(Z), CL/F, V_(z)/F, Ae, fe, and CL_(R).

Analytical Methods

Validated LC-MS/MS detection methods for quantifying plasma and urineconcentrations of 3,4-DAP and its N-acetyl metabolite are described inExample 17a.

Analysis Populations

The following populations were analyzed in the study. The safetypopulation consisted of all subjects who received at least 1 dose ofstudy drug and had at least 1 post dose safety assessment. Thepharmacokinetic population consisted of all subjects who received atleast 1 dose of study drug and had evaluable PK data.

Statistical Methodology

Summary statistics and statistical analysis of the PK data wereperformed for all subjects who received at least one dose of study drugand had evaluable PK data. For continuous data, summary statisticsincluded the arithmetic mean, arithmetic standard deviation (SD),median, minimum, maximum, and N; for log-normal data (e.g. all PKparameters, excluding T_(max), T_(last), and % AUC_(extrap)), thegeometric mean and geometric percent coefficient of variation (CV %) arealso presented. For categorical data, frequency count and percentagesare presented. For the calculation of summary statistics and statisticalanalysis, unrounded data are used.

Baseline measurement was defined as the last non-missing measurementprior to Period 1 study drug administration. Mean change from baselinewas the mean of all individual subjects' change from baseline values.Each individual change from baseline was calculated by subtracting theindividual subject's baseline value from the value at the time point.The individual subject's change from baseline value was used tocalculate the mean change from baseline using a SAS procedure such asProc Univariate. Data analysis was performed using SAS® Version 9.2.

Pharmacokinetic Analysis

AUC_(0-inf), AUC_(0-t), and C_(max) were subject to statistical analysisby fed/fasted status. These PK parameters were log-transformed (base e)prior to analysis and were analyzed using a mixed model. The modelincluded sequence, period, and treatment as fixed effects and subject asa random effect. For these parameters, least squares (LS) means werecalculated for the fed and fasted treatments. Mean differences betweenthe fed and fasted treatments were calculated. The residual variancefrom the mixed model was used to calculate the 90% confidence interval(CT) for the difference between the fed and fasted treatments. Thesevalues were back-transformed to give geometric LS means, a pointestimate, and 90% CI for the ratio of the fed relative to the fastedtreatment.

A food effect on bioavailability was established if the 90% CI for theratio of population geometric means between fed and fasted treatmentswas not contained in the equivalence limits of 80 to 125% forAUC_(0-inf), AUC_(0-t), or C_(max).

Safety Measures

Analysis for the following safety parameters was carried out on theSafety Population: AEs which included all AEs, AEs by severity,drug-related AEs, deaths, SAEs, AEs leading to study discontinuation,and most frequent AEs, standard clinical laboratory tests, vital signs,ECG measurements, routine physical examinations, and prior andconcomitant medications. The cut-off for frequency of AEs reported wasdetermined based on the data. No formal statistical testing wasperformed for the safety analyses; only descriptive statistics wereprovided. Appropriate descriptive statistics for the safety data weredetermined using SAS Version 9.2.

Adverse Events

AEs were coded according to the Medical Dictionary for RegulatoryActivities (MedDRA; Version 13.0). Concomitant medications were codedusing the World Health Organization (WHO; Version March 2009) drugdictionary. The occurrence of AEs was assessed continuously from thetime the subject signed the VID and consent. The reporting period fornon-serious AEs was from the first dose to the final study visit. Theinvestigator determined the severity of each event and the relationshipof an AE to the IP and recorded it on the source documents and AE CRF.The nature, time of onset, duration, severity, and relationship to studydrug was documented, including documentation of investigator assessmentand review. All events were assessed to determine if the AE was serious.

The Investigator determined the severity of each event and the event wasrecorded on the source documents and AE CRF. A coding of “mild” meantthere was no limitation of usual activities, “moderate” meant there wassome limitation of usual activities, “severe” meant there was aninability to carry out usual activities.

Any clinically significant abnormalities found during the course of thestudy were followed up until they returned to normal or could beclinically explained. If a non-serious AE remained unresolved at theconclusion of the study, the investigator, and medical monitor made ajoint clinical assessment as to whether continued follow-up of the AEwas warranted, and the results of this assessment were documented.Resolution was defined as the return to baseline status or stabilizationof the condition with the expectation that it would remain chronic.

The reporting period for AEs that met the criteria of an SAE began fromthe time of signing the VID and consent form through follow-up or earlytermination. An SAE is defined as any AE that meets at least one of thefollowing:

-   -   Results in death.    -   Is life threatening, that is, places the subject at immediate        risk of death from the event as it occurred.    -   Requires in-patient hospitalization or prolongation of an        existing in-patient hospitalization.    -   Results in persistent or significant disability or incapacity.    -   Is a congenital anomaly or birth defect.    -   Is an important medical event that does not meet any of the        above criteria, but may jeopardize the subject or require        medical or surgical intervention to prevent one of the outcomes        listed above.        Clinical Laboratory Assessments

Blood and urine samples were collected for clinical laboratoryevaluations at Screening, prior to dose administration on dosing daysand at the follow-up visit or early termination visit. Additionalclinical laboratory evaluations were performed at other times whenclinically appropriate or if the ongoing review of the data suggested amore detailed assessment of clinical laboratory safety evaluations wererequired. The investigator performed a clinical assessment of allclinical laboratory data and each clinically significant laboratoryresult was recorded as an AE.

The clinical laboratory evaluations performed are listed in thefollowing table.

Clinical Laboratory Evaluations Blood Chemistry Hematology Urine TestsOther Albumin Hemoglobin Appearance Thyroid panel: Alkaline HematocritColor TSH phosphatase WBC count pH Free T3 ALT (SGPT) RBC count Specificgravity Free T4 AST (SGOT) Platelet count Ketones Serology: Directbilirubin Differential Protein HIV antibodies Total bilirubin^(a) cellcount Glucose Hepatitis C antibody BUN Bilirubin HBsAg Calcium NitriteHormonal panel: ^(b, d) Chloride Urobilinogen Estradiol Totalcholesterol Hemoglobin FSH CO₂ Pregnancy hCG Creatinine test ^(c, d)Glucose GGT LDH Phosphorus Potassium Total protein Sodium Uric acid^(a)Direct bilirubin was analyzed only if total bilirubin was elevated.^(b) Analyzed at Screening only and was analyzed at other time points ifconsidered necessary. ^(c) Suspected false positive result resulted inserum hormone panel. ^(d) Females only.Vital Signs

Supine systolic and diastolic blood pressure, supine heart rate,respiration, and oral body temperature were measured at Screening, ≤90min prior to dose, post dose at 0.5, 1, 2, 4, 8, and 24 hours, and atthe Follow-up Visit or Early Termination Visit. Clinically significantchanges from baseline vital signs were reported as AEs.

Electrocardiography

Single, resting 12-lead ECGs with a 10-second rhythm strip wereconducted at Screening, Days 1 and 8 at ≤90 min pre dose andapproximately 30 (±5) min post dose (estimated T_(max)), and when judgedto be clinically appropriate. ECGs were conducted after subjects hadbeen supine for ≥5 min. The ECG machine computed the PR and QTintervals, QRS duration, and heart rate. The QT interval was correctedfor heart rate (QTc) using Bazett's formula (QTcB). A physicianperformed a clinical assessment of each 12-lead ECG. Clinicallysignificant changes from baseline (Day 1 pre dose) were recorded as AEs.

Appropriateness of Measurements

All measurements performed relating to bioavailability of the IPrelative to fed and fasting states were standard measurements. StandardPK and safety assessments were performed.

Example 11 Safety Results from Example 10

Single oral doses of 20 mg amifampridine phosphate were considered to besafe and well tolerated when administered to healthy male and femalesubjects in the fed and fasted state in this study with no SAEs ordiscontinuations related to amifampridine phosphate. The majority of AEsreported were mild in severity and resolved without treatment. In total,50 of the 93 (54%) AEs reported were paraesthesias and these are wellknown side-effects of amifampridine treatment. The incidence of AEs wassimilar between the fed (24 subjects) and fasted (23 subjects) groups,although there was a higher frequency of AEs in the fasted group (61fasted AEs vs. 40 fed AEs). There were no clinically significantfindings in any clinical laboratory evaluations, vital signs, 12-leadECG, or physical examination.

The overall incidence of treatment emergent AEs are summarized byfed/fasted status in the following table.

Summary of Treatment-Emergent Adverse Events by Fed/Fasted Status Numberof Subjects (%) with Adverse Events [Number of Adverse Events] FastedFed Overall (N = 45) (N = 46) (N = 47) Subjects with Adverse 24 (53%) 23(50%) 31 (66%) Events Number of Adverse 61 40 101 Events Subjects withserious adverse events Total 0 (0%) [0]  0 (0%) [0]  0 (0%) [0] Subjects discontinued due to adverse events Total 0 (0%) [0]  1 (2%)[1]  1 (2%) [1]  All treatment emergent adverse events Total 24 (53%)[61] 23 (50%) [40] 31 (66%) [101] Mild 24 (53%) [60] 22 (48%) [38] 30(64%) [98]  Moderate 1 (2%) [1]  1 (2%) [1]  2 (4%) [2]  Severe 0 (0%)[0]  1 (2%) [1]  1 (2%) [1]  Possibly or probably related adverse eventsTotal 21 (47%) [56] 21 (46%) [37] 30 (64%) [93]  Mild 21 (47%) [55] 21(46%) [37] 30 (64%) [92]  Moderate 1 (2%) [1]  0 (0%) [0]  1 (2%) [1] Severe 0 (0%) [0]  0 (0%) [0]  0 (0%) [0]  N, number of subjectsstudied.

The incidence and frequency of treatment emergent AEs are listed byfed/fasted status in the following table.

Incidence of Treatment Emergent Adverse Events by Fed/Fasted Status(Drug-Related) Number of Subjects with Adverse Events [Number of AdverseEvents] System Organ Class Fasted Fed Overall MedDRA Preferred Term (N =45) (N = 46) (N = 47) Nervous System Disorders Total 21 (47%) [43] 17(37%) [31] 27 (57%) [74] Paraesthesia Oral 13 (29%) [15] 13 (28%) [15]20 (43%) [30] Paraesthesia 10 (22%) [12] 7 (15%) [8] 12 (26%) [20]Dizziness 5 (11%) [5] 2 (4%) [2]  5 (11%) [7] Hypoaesthesia Oral 3 (7%)[3]  3 (7%) [3]  5 (11%) [6] Headache 4 (9%) [4]  1 (2%) [1]  5 (11%)[5] Dysgeusia 1 (2%) [1]  2 (4%) [2]  2 (4%) [3]  Hypoaesthesia 2 (4%)[2]  0 (0%) [0]  2 (4%) [2]  Hypoaesthesia Facial 1 (2%) [1]  0 (0%)[0]  1 (2%) [1]  Gastrointestinal Disorders Total  7 (16%) [10] 2 (4%)[2]   9 (19%) [12] Nausea 3 (7%) [3]  1 (2%) [1]  4 (9%) [4]  AbdominalPain 3 (7%) [3]  0 (0%) [0]  3 (6%) [3]  Abdominal Pain Upper 2 (4%)[2]  0 (0%) [0]  2 (4%) [2]  Abdominal Tenderness 1 (2%) [1]  0 (0%)[0]  1 (2%) [1]  Constipation 0 (0%) [0]  1 (2%) [1]  1 (2%) [1] Diarrhoea 1 (2%) [1]  0 (0%) [0]  1 (2%) [1]  General Disorders andAdministration Site Conditions Total 2 (4%) [2]  3 (7%) [3]  5 (11%) [5]Fatigue 1 (2%) [1]  3 (7%) [3]  4 (9%) [4]  Feeling Hot 1 (2%) [1]  0(0%) [0]  1 (2%) [1]  Skin and Subcutaneous Tissue Disorders Total 1(2%) [1]  0 (0%) [0]  1 (2%) [1]  Acne 1 (2%) [1]  0 (0%) [0]  1 (2%)[1]  Vascular Disorders Flushing 0 (0%) [0]  1 (2%) [1]  1 (2%) [1] Total 0 (0%) [0]  1 (2%) [1]  1 (2%) [1]  Overall Total 21 (47%) [56] 21(46%) [37] 30 (64%) [93] MedDRA, Medical Dictionary for RegulatoryActivities; N, number of subjects studied.

The overall incidence of treatment emergent AEs are summarized by genderin the following table. The incidence of AEs was higher in femalesubjects with 12 of 16 subjects (75%) reporting an AE compared to 19 of31 (61%) male subjects.

Summary of Treatment-Emergent Adverse Events by Gender Number ofSubjects (%) with Adverse Events [Number of Adverse Events] Male FemaleOverall (N = 31) (N = 16) (N = 47) Subjects with 19 (61%) 12 (75%) 31(66%) Adverse Events Number of 58 43 101 Adverse Events Subjects withserious adverse events Total 0 (0%) [0]  0 (0%) [0]  0 (0%) [0] Subjects discontinued due to adverse events Total 1 (3%) [1]  0 (0%)[0]  1 (2%) [1]  All treatment emergent adverse events Total 19 (61%)[58] 12 (75%) [43]  31 (66%) [101] Mild 18 (58%) [57] 12 (75%) [41] 30(64%) [98] Moderate 0 (0%) [0]  2 (13%) [2] 2 (4%) [2]  Severe 1 (3%)[1]  0 (0%) [0]  1 (2%) [1]  Possibly or probably related adverse eventsTotal 18 (58%) [51] 12 (75%) [42] 30 (64%) [93] Mild 18 (58%) [51] 12(75%) [41] 30 (64%) [92] Moderate 0 (0%) [0]  1 (6%) [1]  1 (2%) [1] Severe 0 (0%) [0]  0 (0%) [0]  0 (0%) [0]  N, number of subjectsstudied.

Incidence of Treatment Emergent Adverse Events by Gender (Drug-Related)Number of Subjects with Adverse Events System Organ Class [Number ofAdverse Events] MedDRA Male Female Overall Preferred Term (N = 31) (N =16) (N = 47) Nervous System Disorders Total 17 (55%) [43] 10 (63%) [31]27 (57%) [74] Paraesthesia Oral 13 (42%) [18]  7 (44%) [12] 20 (43%)[30] Paraesthesia  6 (19%) [11] 6 (38%) [9] 12 (26%) [20] Dizziness 1(3%) [2]  4 (25%) [5] 5 (11%) [7] Hypoaesthesia Oral 4 (13%) [5] 1 (6%)[1]  5 (11%) [6] Headache 2 (6%) [2]  3 (19%) [3] 5 (11%) [5] Dysgeusia2 (6%) [3]  0 (0%) [0]  2 (4%) [3]  Hypoaesthesia 1 (3%) [1]  1 (6%)[1]  2 (4%) [2]  Hypoaesthesia Facial 1 (3%) [1]  0 (0%) [0]  1 (2%)[1]  Gastrointestinal Disorders Total 4 (13%) [4] 5 (31%) [8]  9 (19%)[12] Nausea 2 (6%) [2]  2 (13%) [2] 4 (9%) [4]  Abdominal Pain 1 (3%)[1]  2 (13%) [2] 3 (6%) [3]  Abdominal Pain Upper 1 (3%) [1]  1 (6%)[1]  2 (4%) [2]  Abdominal Tenderness 0 (0%) [0]  1 (6%) [1]  1 (2%)[1]  Constipation 0 (0%) [0]  1 (6%) [1]  1 (2%) [1]  Diarrhoea 0 (0%)[0]  1 (6%) [1]  1 (2%) [1]  General Disorders and Administration SiteConditions Total 3 (10%) [3] 2 (13%) [2] 5 (11%) [5] Fatigue 2 (6%) [2] 2 (13%) [2] 4 (9%) [4]  Feeling Hot 1 (3%) [1]  0 (0%) [0]  1 (2%) [1] Skin and Subcutaneous Tissue Disorders Total 1 (3%) [1]  0 (0%) [0]  1(2%) [1]  Acne 1 (3%) [1]  0 (0%) [0]  1 (2%) [1]  Vascular DisordersFlushing 0 (0%) [0]  1 (6%) [1]  1 (2%) [1]  Total 0 (0%) [0]  1 (6%)[1]  1 (2%) [1]  Overall Total 18 (58%) [51] 12 (75%) [42] 30 (64%) [93]MedDRA, Medical Dictionary for Regulatory Activities; N, number ofsubjects studied.

Study drug-related AEs in the nervous system disorders system organclass had the highest incidence (27 subjects; 57%) and frequency (74events). The second highest incidence (9 subjects; 19%) and frequency(12 events) of AEs was in the gastrointestinal disorder system organclass. The most frequently reported AEs considered as being drug-relatedwere paraesthesias; oral paraesthesia (30 events) and paraesthesia (20events); dizziness (7 events); oral hypoaesthesias (6 events); headache(5 events); fatigue (4 events); nausea (4 events); dysgeusia (3 events)and abdominal pain (3 events). Hypoaesthesia and upper abdominal painwere the only other AEs possibly related to amifampridine treatment thatwas reported by more than 1 subject in the study.

In total, 50 (54%) of the 93 AEs were paraesthesias and these are wellknown side effects of amifampridine treatment. All reported incidencesof paraesthesia and hypoaesthesia were mild and were considered aspossibly related to the administered treatment by the investigator.Overall, most drug-related AEs were transitory and mild, and allsubjects recovered from the AE.

The incidence of drug-related AEs was identical in the fed and fastedgroups (21 subjects). However, the frequency of AEs was higher in thefasted group (56 events) than the fed (37 events). Oral paraesthesia wassimilarly prevalent in the fed and fasted groups; paraesthesia was morefrequently reported in the fasted group (12 events) than the fed (8events). Incidence and frequency of oral hypoesthesia was identical inthe fed and fasted groups (3 events). Hypoaesthesia (2 events) andfacial hypoesthesia (1 event) were only reported by subjects in thefasted group.

Nervous system disorders had a higher incidence and frequency in thefasted state (21 subjects; 43 events) than in the fed (17 subjects, 31events) state. In addition to paresthesias, headache and dizziness weremore common in the fasted state. Dysguesia was the only adverse eventmore frequent in the fed state. Gastrointestinal disorders had a higherincidence and frequency in the fasted state (7 subjects; 10 events) thanthe fed (2 subjects; 2 events). Abdominal pain, nausea, upper abdominalpain, abdominal tenderness and diarrhea were all more commonly reportedby subjects following administration of the study drug in the fastedstate.

In general, there was a higher incidence of drug-related AEs in females(75%) than males (58%). Incidences of peripheral paraesthesia werehigher in female subjects (38%) than males (19%), and gastrointestinaldisorders were also more common in females (31%) than males (13%).

Only 1 AE of moderate severity was reported during the study as beingpossibly related to the study drug. One subject experienced an AE ofheadache in Period 2 (fasted treatment) 4 and a half hours after studydrug administration. The AE lasted for approximately 8 hours andrequired treatment with one oral dose of paracetamol (1 g).

There were no deaths or SAEs during this study.

Example 12 Pharmacokinetics Results from Example 10

Pharmacokinetics of Amifampridine Following Administration in the Fastedand Fed State

Mean plasma concentrations (+SD) of Amifampridine and 3-N-AcetylAmifampridine of patients in fed state following oral administration ofAmifampridine Phosphate are given in FIG. 6. Mean plasma concentrations(+SD) of Amifampridine and 3-N-Acetyl Amifampridine of patients infasted state following oral administration of Amifampridine Phosphateare given in FIG. 7. 3,4-DAP Plasma Levels at 4 Hours Post Dose forSubjects in Fasted State are given in FIG. 8. FIG. 8 depicts 3,4-DAPplasma levels at 4 hours post dose suggesting an emerging bimodaldistribution by plasma concentration in fasted subjects. Subjects with aplasma concentration of 3,4-DAP phosphate between 0 and 5 ng/mL arelikely to be fast acetylators. Subjects with a plasma concentration of3,4-DAP phosphate between 10 and 26 ng/mL are likely to be slowacetylators. PK for two patients after a single oral dose of 3,4-DAP ina fasted state is given in FIG. 9.

The mean plasma amifampridine concentration-time profiles for fed andfasted administrations are shown in FIG. 10, indicating that C_(max) isdampened when amifampridine is administered with food. The overall meanPK parameter values for amifampridine exposure (C_(max), AUC_(0-t),AUC_(0-inf), T_(max), t_(1/2), and λ_(z)) in fed and fasted states aregiven in FIG. 11. The mean C_(max) was higher in the fasted state, aswere the mean values for AUC_(0-t) and AUC_(0-inf). Mean t_(1/2) wassomewhat longer, and inversely, the terminal elimination rate constantλ_(z) was somewhat shorter in the fasted state. Mean T_(max) was shorterin the fasted state compared with the fed state.

Variability in amifampridine mean PK parameters for C_(max)(CVs rangingfrom 58.2% to 77.1%) and AUC was high (FIG. 11). The high variabilitywas also apparent in the wide individual ranges which tended to spanmore than 10-fold for AUC_(0-t), AUC_(0-inf) and C_(max) in either thefasted or fed state. Specifically, the individual subject data forAUC_(0-t) ranged from 20.6 to 267 ng·h/mL in the fasted state, and from8.3 to 282 ng·h/mL for the fed state; and C_(max) ranged from 16.0 to137 ng/mL in the fasted state and from 2.81 to 132 ng/mL in the fedstate. There was less variability in t_(1/2) and λ_(z), which had CVsbetween 29.2% and 39.0%. Specifically, the individual subject data fort_(1/2) ranged from 1.23 to 4.31 hours in the fasted state and from0.822 to 3.78 hours in the fed state.

The statistical comparison of pharmacokinetic parameters foramifampridine phosphate is summarized in the following table.

Statistical Comparison of Pharmacokinetic Parameters for AmifampridinePhosphate after Oral Administration of 20 mg of Amifampridine to HealthySubjects in Fed/Fasted State

Statistical Comparison of Pharmacokinetic Parameters for AmifampridinePhosphate After Oral Administration of 20 mg of Amifampridine to HealthySubjects in Fed/Fasted State Geometric Mean Ratio (%)^(a) Fed/Fasted Fedvs. Fasted 90% Confidence Interval parameter Point Estimate Lower limitUpper Limit C_(max) 56.3 47.0 67.5 AUC_(0-t) 80.0 73.1 87.6 AUC_(0-inf)82.3 76.0 89.2 ^(a)Based on analysis of natural-log transformed data.AUC_(0-t), area under the plasma concentration-time curve from time zeroup to the last measurable concentration; AUC_(0-inf), area under theplasma concentration-time curve from time zero to infinity; C_(max)maximum observed plasma concentration

The extent of exposure based on AUC with geometric mean ratios(fed/fasted) of 80.0% (AUC_(0-t)) and 82.3% (AUC_(0-inf)) indicates thatexposure to amifampridine is decreased approximately 18 to 20% followingadministration in fed vs. fasted state. The C_(max) geometric mean ratio(fed/fasted) of 56.3% indicates that the maximum plasma concentrationwas decreased 44% in the presence of food. The associated 90% confidenceintervals are 47.0 to 67.5% for C_(max), 73.1 to 87.6% for AUC_(0-t),and 76.0 to 89.2% for AUC_(0-inf), which are all outside of theallowable 80 to 125% equivalence range, indicating an effect of food onamifampridine exposure.

Pharmacokinetics of 3-N-Acetyl Amifampridine Following Administration inthe Fasted and Fed States

The main plasma metabolite of amifampridine is 3-N-acetyl amifampridine.The concentration-time profiles for appearance of the metabolite in thefed and fasted states are shown in FIG. 12. As observed withamifampridine, the metabolite reached higher C_(max) levels with shorterT_(max) in the fasted state compared to the fed state. The mean PKparameter values for 3-N-acetyl amifampridine exposure (C_(max),AUC_(0-t), AUC_(0-inf), T_(max), t_(1/2) and λ_(z)) in fed and fastedstates are given in FIG. 13.

The maximal mean plasma concentration (C_(max)) of 3-N-acetylamifampridine was almost 1.5-fold higher in the fasted state compared tothe fed state, though there was considerable overlap in the range ofdata observed in individual subjects. Mean plasma T_(max) was almost1.7-fold longer in the fed state, but individual data values ranged from0.75 to 4.0 hours in the fed state and 0.75 to 2.0 hours in the fastedstate. Both mean AUC_(0-t) and AUC_(0-inf) for 3-N-acetyl amifampridinewere fairly comparable in the fed and fasted states. Overall CVs for PKparameters for the metabolite were smaller than those for the parentcompound. Comparison of the mean plasma concentrations of amifampridineand 3-N-acetyl amifampridine in fed and fasted states are shown in FIGS.6 and 7.

The mean concentration of the major metabolite, 3-N-acetylamifampridine, was greater than that of amifampridine in plasma in allsubjects. Mean metabolite exposure indices were calculated based onratios of PK parameters for metabolite:parent drug (i.e., 3-N-acetylamifampridine:amifampridine). For C_(max), the ratio was 4.53 in thefasted state and 4.65 in the fed state, indicating that the maximalconcentration of metabolite is approximately 4.5-fold higher than thatof parent compound despite influence of food on C_(max) For meanAUC_(0-t), the ratio was 11.6 in the fed state and 12.5 in the fastedstate; for mean AUC_(0-inf), 11.2 in the fed state and 12.3 in thefasted state. Thus regardless of effects of food, exposure to metaboliteis about 12-fold greater than that to parent compound. The mean plasmaelimination t½ for the 3-N-acetyl metabolite (4.03 hours fed; 4.10 hoursfasted) was longer than that for amifampridine (2.28 hours fed; 2.50hours fasted).

Urinary Excretion

Urinary excretion of amifampridine and 3-N-acetyl amifampridine in fedand fasted subjects is shown in FIG. 14. Urinary excretion dataindicated that the fraction of unchanged amifampridine eliminated in the0 to 24 h urine collection averaged 18.8% (fasted) to 19.2% (fed) of theadministered dose. The 3-N-acetyl metabolite was also extensivelyeliminated in the 0 to 24 h urine and represented 74.0% (fed) to 81.7%(fasted) of the administered dose. The total of parent drug andmetabolite eliminated in the 0 to 24 h urine represented 93.2% (fed) to100% (fasted) of the administered dose. Together these results indicatethat a single oral dose of amifampridine is essentially completelyexcreted in the urine over a 24-hour period as unchanged drug (˜19%) anda single major metabolite (˜75-80%).

Pharmacokinetic Conclusions

The plasma concentrations of amifampridine and its major metabolite(3-N-acetyl amifampridine) were quantified out to 24 hours in bothplasma and urine after single oral doses of 20 mg amifampridinephosphate. Statistically, comparison of key PK parameters (C_(max) andAUC) between the fed and fasted states did not establish comparability.Specifically, the 90% Cl of the geometric mean ratios of fed:fasted were47.0 to 67.5% for C_(max), 73.1 to 87.6% for AUC_(0-t), and 76.0 to89.2% for AUC_(0-inf). These values fall outside of the standardequivalence limits of 80 to 125%, indicating that there is a significanteffect of food on amifampridine exposure.

Overall there is a decrease in exposure of ˜40% in C_(max) and ˜20% inAUC due to oral administration of amifampridine phosphate in thepresence of food. Mean T_(max) is shorter in the fasted state (0.637hours) than in the fed state (1.31 hours). Together these findingssuggest that food slows and decreases absorption of amifampridinephosphate. The delay in T_(max) may be attributed to differences ingastric emptying times in the presence of food delaying the introductionof drug to the duodenum where absorption occurs.

This is the first human study measuring the major metabolite, 3-N-acetylamifampridine, in addition to the parent compound. 3-N-acetylamifampridine levels were higher than amifampridine in all subjects,whether fed or fasted. In both the fed and fasted states, C_(max) for3-N-acetyl amifampridine was approximately 4.5-fold higher and AUCvalues were approximately 12-fold higher than those for amifampridine.The mean plasma t_(1/2) for the 3-N-acetyl amifampridine was longer thanthat for amifampridine in both the fed state (4.03 hours for 3-N-acetylamifampridine vs. 2.28 hours for amifampridine), and fasted state (4.10hours for 3-N-acetyl amifampridine vs. 2.50 hours for amifampridine).Thus duration and extent of exposure to the metabolite is greater thanthat of the parent compound. At present, preliminary in-vitro results incloned K+ channel test systems indicate that the 3-N-acetylamifampridine metabolite most likely does not inhibit K+ channels up toits solubility limit in the test system.

Urinary excretion data indicate that the fraction of unchangedamifampridine eliminated in the 0 to 24 hour urine collection averaged19% of the administered dose. The 3-N-acetyl metabolite was extensivelyeliminated in the 0-24 h urine and represented 74.0% to 81.7% of theadministered dose. The mean total of parent drug and metaboliteeliminated in the 0-24 h urine represented 93.2% to 100% of theadministered dose. Orally administered amifampridine appears to beessentially completely eliminated from the body within 24 hours as theparent compound and a single metabolite.

High inter-individual variability was observed in this food-effectstudy. The inter-individual CVs for C_(max) and AUC are ˜60-70%, withparameters for individual subjects varying over more than a 10-foldrange. While acetylator status was not determined in this study,Applicants hypothesized and later demonstrated that the most probableexplanation for this variability is the metabolic disposition ofamifampridine through a single polymorphic metabolic pathway viaN-acetyltransferase enzymes (NAT1 and NAT2). NAT enzymes are recognizedto be highly polymorphic in the human population, with recognized slowand fast acetylator phenotypes. It is likely that acetylator statusaffects the overall PK and disposition of amifampridine.

Discussion and Overall Conclusions

This study investigated the relative bioavailability of a single oraldose of 20 mg amifampridine phosphate when administered to humans in thefed compared to the fasted state. Statistical comparison of keyparameters (i.e., C_(max) and AUC) between the fed and fasted states didnot establish PK equivalence. Overall there was a decrease in exposureof ˜40% in C_(max) and ˜20% in AUC when amifampridine phosphate wasadministered in the presence of food. Observed differences could be dueto decreased or delayed absorption at the level of the duodenum fromchanges in gastric motility (Rowland and Tozer, ClinicalPharmacokinetics, Concepts and Applications, 3^(rd) Edition, 1995, Ch 9,Lippincott Williams and Wilkins Publishers). Although the mean C_(max)and AUC PK parameter differences between fed and fasted states arestatistically significant, they are less than the variability in theseparameters observed between individual subjects within eitheradministration state. While acetylator status was not determined in thisstudy, Applicants show in later studies that this variability is due tothe metabolic disposition of amifampridine through a single pathway viaNAT enzymes. NAT is recognized to be highly polymorphic in the humanpopulation (Hiratsuka M, Kishikawa Y, Tukemura Y, et al. Genotyping ofthe N-acetyl transferase 2 polymorphism in the prediction of adversedrug reactions to Isoniazid in Japanese patients. Drug Metab Pharmacokin2002; 17; 357-362), with recognized slow and fast acetylator phenotypes.For instance, 50-59% of the Caucasian population are slow acetylators,while 41-50% are rapid acetylators (Casarett & Doull's Toxicology, TheBasic Science of Poisons 7th Ed. (2008) Chapter 6: Biotransformation ofXenobiotics. pp. 278-282). It is possible that acetylator status affectsthe overall PK and disposition of amifampridine.

Renal excretion accounted for greater than 94% of the orallyadministered dose eliminated within 24 hours and was represented byunchanged parent drug (˜19%) and the 3-N-acetyl metabolite (75-89%).This indicates that there are unlikely to be any other quantitativelysignificant metabolites and that an oral dose of amifampridine israpidly excreted. In addition, it suggests that dosage adjustments maybe required in patients with renal impairment.

The 3-N-acetyl amifampridine metabolite plasma concentration levels werehigher than amifampridine in all subjects in either the fasted or fedstates. Overall, AUC exposure to the metabolite was approximately12-fold higher compared to that of the parent drug.

The metabolite is not predicted to bind or fit into the K+ ion channel.In-vitro studies to evaluate interaction with several K+ ion channelsare described in Examples 5 and 6. In fact, the N-acetyl metabolite isnot active in several potassium ion channels. (See FIGS. 20, 21 a, and21 b.)

Single oral doses of 20 mg amifampridine phosphate were considered to besafe and well tolerated when administered to healthy subjects in the fedand fasted conditions of this study. The majority of AEs reported weremild in severity and resolved without treatment. Only one severe AEresulting in subject withdrawal was reported during the study: anepisode of gastroenteritis which was not considered to be related to thestudy drug by the investigator. There were no SAEs reported during thestudy. The most frequent AEs were paresthesias, which are well knownside effects of amifampridine treatment. The incidence of AEs, in termsof the number of subjects reporting AEs, was similar between the fed andfasted groups, although in terms of number of AEs, there was a higherfrequency of AEs in the fasted group (61 fasted, 40 fed).

The variability of amifampridine PK is expected to translate intovariations in clinical safety or efficacy and indeed Applicantsdemonstrated in later examples that frequency of adverse events iscorrelated to a patient's acetylator status. Efficacy was not evaluatedin this study. Despite the range of observed amifampridine and3-N-acetyl metabolite levels, no related severe or serious AEs wereobserved in this study. However, more moderate adverse events may bereduced in severity or frequency by determining a patient's acetylationstatus and dosing amifampridine with or without food as appropriate.

In summary, oral administration of amifampridine phosphate with food hasa statistically significant lowering effect on maximal concentration andoverall exposure. Amifampridine phosphate was demonstrated to be rapidlyabsorbed, extensively metabolized, and essentially completely eliminatedwithin 24 hours by the renal route. Overall, these data provideadditional information on the bioavailability, PK, and safety ofamifampridine phosphate that potentially inform the safe and effectiveuse of amifampridine phosphate in patients.

Example 13 Preparation of 3,4-Diaminopyridine Phosphate

A variety of methods are known in the art for synthesis of3,4-diaminopyridine, precursors, derivatives and analogs. In addition,it is commercially available from sources such as VWR, ASINEX, andMaybridge. 3,4-DAP phosphate used in the experiments in this applicationwere prepared using procedures similar to or the same as those describedin US20040106651 which is included below.

Stage 1) Synthesis of 3,4-DAP Phosphate: 90 parts of 3,4-DAP (Aldrich),purified beforehand, and 1800 parts of distilled water are introducedinto a reactor. The mixture is brought to a temperature of 75° C. withstirring. Dissolution is observed to be complete. Subsequently, 191parts of 85% phosphoric acid are slowly introduced into the 3,4-DAPsolution. After the addition of the phosphoric acid, the reactionmixture is kept at a temperature of 80° C. for a further 15 min and isthen cooled to 35° C. The reaction mixture is then kept at a temperatureof between 30 and 35° C. for 4 hours with stirring. The precipitateformed is drained and washed with 100 parts of distilled water and thenwith 100 parts of absolute ethanol. After drying under vacuum at 60° C.to constant weight, 160 parts of crude 3,4-DAP phosphate are obtained inthe form of a white powder, the melting point of which is between 225and 227° C.

Stage 2) Purification of the Crude 3,4-DAP Phosphate: 160 parts of crude3,4-DAP phosphate obtained above in stage (1), 640 parts of absoluteethanol and 715 parts of distilled water are introduced into a reactor.The mixture is heated, with stirring, to a temperature of 80° C. At thistemperature, dissolution is complete. The reaction mixture issubsequently cooled gradually to a temperature of 4° C. and is held atthis temperature for 12 hours with stirring. After draining and washingwith 100 parts of absolute ethanol, 180 parts of wet product areobtained. The product is subsequently dried at 60° C. under vacuum toconstant weight. 133 parts of 3,4-DAP phosphate are then obtained, themelting point of which is 229° C. The elemental analysis of the productthus obtained was carried out on a Perkin-Elmer CHN 4000 device. Theproduct sample was weighed on a balance with an accuracy of 10.sup.-4mg; the percentage of oxygen was calculated by difference. The elementalanalysis of the product obtained, in accordance with that of theexpected product, was as follows

% C H N P O Calculated 28.99 4.83 20.29 14.97 30.92 Found 29.05 4.9320.23 not determined not determined

Example 14 3,4-Diaminopyridine Phosphate Tablet Formulation

Each tablet contained 3,4-diaminopyridine phosphate (equivalent to 10 mgof 3,4-diaminopyridine free base). Each tablet contained about 7.6%(w/w) amifampridine phosphate, about 89.9% (w/w) microcrystallinecellulose, about 0.5% (w/w) colloidal silicon dioxide, and about 2%(w/w) calcium stearate. Acceptable ranges (% weight/weight) are asfollows: 3,4-diaminopyridine, 7.00-13.00; microcrystalline cellulose,89.00-91.00; colloidal silicon dioxide, 0.01-1.00; and calcium stearate,1.00-3.00.

Example 15 NAT2 Phenotypic Assays

A subject's N-acetyltransferase 2 (NAT2) phenotype can be determined ina number of ways to determine whether the subject is a fast or slowacetylator. The examples given in Examples 15 and 15a, and throughoutthe specification are exemplary and are not meant to be limiting.

Whether a person has a slow or fast metabolic phenotype can be assessedusing ratios of urinary caffeine metabolites. Methods are described inCascorbi et al, Am. J. Hum. Genet., 57:581-592, 1995 (“Cascorbi”) andJetter et al, Eur J Pharmacol (2009) 65:411-417 both of which areincorporated herein by reference in their entireties.

Cascorbi discloses the following method. A patient is given one cup ofcoffee or half a tablet of caffeine (Coffeinum 0.2 g compretten N).Urine is collected for 5 hours and the pH is adjusted to 3.5 with 80mmol/L citric acid/phosphate buffer. The prepared urine can be stored at−20° C. until analysis is done. To 0.2 mL of urine saturated with 120 mgammonium sulfate is added 6 mL chloroform/2-propanol (95:5 v/v) and 0.2mL chloroform containing 24 μg N-acetyl-4-aminophenol, for example, asan internal standard. The sample is then shaken and centrifuged and theorganic phase is lyophilized and resuspended in 1 mL 0.05% acetic acid.The sample is then analyzed by HPLC for the presence of5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylxanthine(1X). This can be done on a Beckman ultrasphere octadecylsilane HPLCcolumn with 4 mm internal diameter and 25 cm length and 5 μm particlesize (or an analogous column). The detector wavelength is set at 280 nm,the eluent is 0.05% acetic acid/methanol (92:8 v/v), and the flow rateis 1.2 mL/minute (or can be done using analogous conditions). Forcalibration 1X and AFMU can be added to blank urine samples. Acetylationphenotype is then evaluated by the molar ratio of log (AFMU/1X) where aresult of at or below about −0.30 means the person is a slow metabolizerand a result above about −0.30 means the person is a fast metabolizer.

Alternatively, the analytical method described in Jetter et al. Eur JClin Pharmacol 2004, 60:17-21 can be used.

Alternatively, acetylation phenotype can be determined according to theEidus method using isoniazid as a model drug (Eidus et. al. Bull WorldHealth Organ 1973, 49, 507-516; Rychlik-Sych et. al. PharmacologicalReports 2006, 58, 2-29). Isoniazid is administered orally at a singledose of 10 mg/kg of body weight. Urine samples are collected 6-8 hoursafter drug administration. Isoniazid and acetylated isoniazidconcentrations are determined spectrophotometrically at 550 nmwavelength. The ratio of extracted acetylisoniazid to a total amount ofthe extracted isoniazid in urine is calculated as follows: A/(B−A)×0.761where A is the optical density of an aliquot containing onlyacetylisoniazid; B is the optical density of an aliquot containingacetylisoniazid and isoniazid artificially converted to acetylisoniazid;and multiplication by 0.761 compensates for the molecular weight ofacetylisoniazid and free isoniazid. A value below 3 characterizes a slowacetylator, above 5 as a fast acetylator, and between 3 and 5 as anintermediate acetylator (but is generally categorized as a fastmetabolizer).

Alternatively, administration of sulfamethazine, hydralazine, phenytoin,sulfadiazine, and procainamide can be used to determine a person'sacetylation status.

Alternatively, a subject's phenotype can be determined using methodsdescribed in Br J Clin Pharmacol. 2000, 49(3), 240-243 or Archives ofToxicology 2005, 79(4), 196-200. A person of ordinary skill in the artwould know other methods that could be used to determine a person's NAT1and NAT2 phenotype.

Example 15a NAT2 Phenotypic Assay: Caffeine Test Used in Example 18

Phenotyping can be accomplished using the following method (Schneider,et. al. J. Chromatog. B 2003, 789, 227-237). For the trial in Example18, subjects emptied their bladders immediately before theadministration of a single oral dose of 150 mg caffeine in 240 mL tapwater. Subjects had fasted for at least four hours before dosing andfasted for at least one hour after dosing. All urine was collected for a6 hour period after dosing. Samples were frozen and stored at −20° C.until analysis. The urinary concentration of 1X, AAMU, and 1U werequantified by LC-MS/MS on an Acquity HSS T3 column (100×2.1 mm, 1.8 μm,at 40±1° C., Waters). A mixture of 98% mobile phase A (0.1% formic acidin water) and 2% mobile phase B (methanol) was used to elute themetabolites. A subject's acetylator phenotype was determined accordingto the following equation (“ratio 1”): (AFMU+AAMU)/(AFMU+AAMU+1X+1U).AFMU was not quantified. Instead it was converted before sampleprocessing to AAMU. In general fast acetylators have a ratio of greaterthan about 0.2, in another example between about 0.2 to about 0.3, inanother example greater than 0.3, in another example between about 0.3and about 0.6; and slow acetylators have a ratio of about 0.2 or less,for example between about 0.1 and about 0.2 (inclusive). The subjects inthe trial described in Example 18 were phenotyped using this method.Results are given in Table 5 below in Example 16.

Example 16 NAT2 Genotypic Determination

N-acetyltransferase 2 (NAT2) genotype for a subject can be identified ina number of ways to determine whether a subject is a fast or slowacetylator. The examples given herein and throughout the specificationare exemplary and are not meant to be limiting. In particular, differentpopulations of people may have different mutations which were notdisclosed here, but are known to one of ordinary skill in the art. Forexample, a person's genotypic status can be determined using methodsknown in the art, for example Cascorbi et al, Am. J. Hum. Genet.,57:581-592, 1995 which is incorporated herein by reference in itsentirety.

Additionally, genotyping can be accomplished using the following method.For the trial in Example 18, a 10 mL sample of blood was collected fromeach subject in an K₂EDTA tube. Samples were stored at 4° C. for amaximum of five days. If stored for longer, they were stored at −20° C.Genomic DNA was isolated from EDTA-anticoagulated peripheral blood usingstandard methods. Genomic DNA was placed in a 96-well plate andtransferred to a StepOnePlus Real Time PCR system from AppliedBiosystems (TaqMan SNP genotyping with allelic discrimination). Theback-ground fluorescence signal was determined before amplification.Using PCR, the DNA sequence concerned was labeled with the molecularprobes C282T and T341C and was surrounded by primers. The NAT 2 geneswere exponentially amplified and the fluorescent markers incorporated.The molecular probes C282T and T341C detect about 95% of all NAT2mutations. For NAT2 T341C, the Wild Type allele is identified with theFAM™-labeled probe and the mutant with the VIC®-labeled probe. For NAT2C282T, tie Wild Type allele was identified with the VIC®-labeled probeand the mutant with the FAM™-labeled probe. A person with at least onefast alleles was categorized as a fast acetylator. A person with no fastalleles was categorized as a slow acetylator. A summary of possible NAT2gene polymorphisms is outlined in the Table 4 below using two molecularprobes: C282T and T341C. Genotyping and phenotyping results for eachsubject in Example 18 are given in Table 5.

TABLE 4 Possible NAT2 gene polymorphisms T341C C282T Assigned PhenotypeStatus Wildtype Wildtype Rapid Wildtype Heterozygote IntermediateWildtype Mutant Slow Heterozygote Wildtype Intermediate HeterozygoteHeterozygote Slow or Intermediate depending on location (caffeine)Heterozygote Mutant Slow Mutant Wildtype Slow Mutant Heterozygote SlowMutant Mutant Slow

TABLE 5 Genotyping and phenotyping results per patient Ratio 1, SubjectGender Ex. 15a Phenotype T341C C282T Genotype Part 1, Group 1 1 M 0.457fast Wildtype Heterozygote fast 2 F 0.428 fast Heterozygote Wildtypefast 3 M 0.154 slow Heterozygote Heterozygote slow 4 M 0.135 slowWildtype Homozygote slow mutant 5 F 0.409 fast Heterozygote Wildtypefast 6 M 0.380 fast Wildtype Heterozygote fast 7 M 0.182 slow WildtypeHomozygote slow mutant 8 M 0.189 slow Homozygote Wildtype slow mutant 9F 0.378 fast Heterozygote Wildtype fast 10 M 0.394 fast WildtypeHeterozygote fast 11 F 0.194 slow Wildtype Homozygote slow mutant 12 F0.176 slow Homozygote Wildtype slow mutant Part 2, Group 1 1 M 0.465fast Wildtype Heterozygote fast 2 F 0.433 fast Wildtype Heterozygotefast Part 1, Group 1 3 M 0.154 slow Heterozygote Heterozygote slow 4 M0.195 slow Heterozygote Heterozygote slow Part 2, Group 2 1 M 0.150 slowHeterozygote Heterozygote slow 2 M 0.146 slow Heterozygote Heterozygoteslow 3 M 0.152 slow Heterozygote Heterozygote slow 4 M 0.195 slowWildtype Homozygote slow mutant 5 M 0.154 slow Wildtype Homozygote slowmutant 6 F 0.456 fast Heterozygote Wildtype fast 7 F 0.407 fast WildtypeWildtype fast 8 M 0.515 fast Wildtype Wildtype fast 9 M 0.415 fastWildtype Heterozygote fast 10 M 0.393 fast Heterozygote Wildtype fast

Other NAT2 gene mutations are induced by replacement of a wild typeallele nucleotide sequence at positions 192, 282, 341, 434, 481, 590,813, 845, and 857. These mutations cause impaired acetylation. Inparticular, 4 mutations—191A, 481T, 590A, and 857A—account for nearlyall slow acetylator alleles among blacks, whites, Asian Indians,Koreans, Japanese, Hong Kong Chinese, Taiwanese, Filipinos, and Samoans(Lin et. al. Pharmacogenetics 1994, 4(3), 125-134). A subject can betested for these mutations in order to determine whether they are slowacetylators.

The NAT 1 gene can be examined for 7 single nucleotide polymorphisms(SNPs) in alleles *3, *10, *11, *14, *15, and *17.

Alternatively, a subject's genotype can be determined using methodsdescribed in Br J Clin Pharmacol. 2000, 49(3), 240-243 or Archives ofToxicology 2005, 79(4), 196-200. A person of ordinary skill in the artwould know other methods that could be used to determine a person's NAT1and NAT2 genotype.

Example 17a LC/MS/MS Determination of 3,4-Diaminopyridine (3,4-DAP) inHuman Plasma and Urine

The following method was used to analyze concentrations of3,4-diaminopyridine and N-(4-aminopyridin-3-yl)acetamide in plasma andurine samples for the experiments in Example 10. In the LC/MS/MS method,the human plasma and urine samples were extracted using acetonitrilecontaining 0.1% formic acid. Samples were analyzed by using normal-phaseHPLC with Turbo-Ion Spray® MS/MS detection. The column was an AtlantisHILIC Silica column (3 μm, 2.1×100 mm) at a temperature of 45° C. Mobilephase A was acetonitrile/isopropanol (90/10; v/v) containing 0.1% formicacid. Mobile phase B was water containing ammonium formate (20 mM) and0.1% formic acid. The flow rate was 800 μL/minute. The gradientconditions for human plasma and urine samples were as follows:

Time (min) A (%) B (%) 0.0 95 5 0.5 95 5 4.0 90 10 4.5 20 80 5.5 20 805.6 95 5 7.0 95 5The LC/MS/MS method was validated to quantify 3,4-DAP in human plasma inthe linear calibration range of 0.5 to 500 ng/mL and from 2 to 2000ng/mL for the 3-N-acetyl metabolite. The validated method used for urinequantification ranged from 150 to 15000 ng/mL for amifampridine and the3-N-acetyl metabolite.

Example 17b LC/MS/MS Determination of 3,4-Diaminopyridine (3,4-DAP) inHuman Plasma and Urine

The following method was used to analyze concentrations of3,4-diaminopyridine and N-(4-aminopyridin-3-yl)acetamide in plasma andurine samples for the experiments in Example 18. In the LC/MS/MS method,the human plasma and urine samples were extracted using acetonitrilecontaining 0.1% formic acid. Samples were analyzed by using HPLC withpositive ion electrospray MS/MS detection. The column was an AtlantisHILIC Silica column (3 μm, 3×50 mm) at a temperature of 40° C. Mobilephase A was water containing ammonium formate (20 mM) and 0.1% formicacid. Mobile phase B was acetonitrile/isopropanol (90/10; v/v)containing 0.1% formic acid. The flow rate was 0.90 mL/minute. Thegradient conditions for human plasma and urine samples were as follows:

Time (min) A (%) B (%) 0.00 10 90 0.50 10 90 2.00 30 70 2.80 90 10 3.6090 10 3.70 10 90The LC/MS/MS method was validated to quantify 3,4-DAP in human plasma inthe linear calibration range of 0.500 to 500 ng/mL and from 1.00 to 1000ng/mL for the 3-N-acetyl metabolite.

Example 18 Clinical Trial to Study the Safety of Firdapse on Slow andFast Acetylators

Objectives

The primary objective of the study was to assess the safety andtolerability of 3,4-DAP phosphate after single and multiple doses. Thesecondary objectives of the study were

-   -   Part 1 only: To determine the dose-related PK profile of        amifampridine and of 3-N-acetyl amifampridine after single,        escalating doses of 3,4-DAP phosphate    -   Part 2 only: To assess the steady-state plasma PK and        accumulation of amifampridine and of 3-N-acetyl amifampridine        over multiple days of dosing with 3,4-DAP phosphate        An exploratory objective was to examine the overall activity of        the N-acetyl transferase enzymes on the metabolism of        amifampridine by assessing the PK of amifampridine and        3-N-acetyl amifampridine with phenotypic acetylation activity        and genotyping determination of NAT2 gene polymorphisms.        Study Design and Plan

This open-label study in healthy subjects (male and female) wasconducted in 2 parts. A total of 26 healthy male and female subjects, 12in Part 1 (at least 5 of each sex), 4 in Part 2 Group 1, and 10 in Part2 Group 2 (at least 4 of each sex) were enrolled into the study. Thesame subjects were not enrolled to participate in both parts. Subjectsin Part 2 were dosed only after all subjects of Part 1 completed dosingand at least 24-hours of follow-up safety evaluation after the last dosewere completed.

All subjects were characterized for their metabolic acetylationphenotype and NAT2 genotype in order to divide subjects into two groupsof acetylators. Volunteers were genetically typed for highly variantalleles of the N-acetyl transferase 2 enzyme (NAT2) domain on chromosome8 (according to Example 16) and were phenotyped for acetylation rateswith a caffeine challenge test (according to Example 15a). Approximatelyequal numbers of slow and fast acetylators (as defined by a caffeinechallenge test in Example 15a) were enrolled in each group. In Part 2,very slow acetylators with NAT2 activity (ratio 1, Example 15a) below0.06 were excluded from participation in the study. In each part of thestudy, subjects were assessed for PK, safety, and tolerability and alldoses were administered within 30 min of ingestion of a meal or a snack.

Part 1

Part 1 was a dose-proportionality study in 12 healthy subjects eachreceiving 4 ascending single oral doses of 3,4-DAP phosphate in asequential manner (5 mg, 10 mg, 20 mg, and 30 mg). For all 12 subjects,the first 3 doses of oral 3,4-DAP phosphate (5-20 mg) were administeredon 3 consecutive days (Days 1-3). Two fast and two slow acetylators (asdefined by a caffeine challenge test in Example 15a) were selected asthe first 4 enrolled subjects (sentinel subjects). For the 2 fastacetylators the 30 mg dose was administered on Day 4. No seizures wereseen within 24 hours of dosing and thus the remaining 2 slow acetylatorswere dosed on Day 5. No seizures were seen in the two slow acetylatorsdosed on Day 5. Safety and tolerability data collected for all 4subjects were reviewed and the safety and tolerability results of the 30mg dose for these 4 sentinel subjects were acceptable. The 30 mg dosewas given to the remaining 8 non-sentinel subjects on Day 7. Sentineland non-sentinel subjects were released from the clinic on Day 5, Day 6,and Day 8 respectively, with all subjects returning for follow-upassessments 2-4 days following their last dose. See Table 1.

TABLE 1 Part 1 Cohort 3,4-DAP Number phosphate Firdapse ® of SubjectsDose (mg base Tablets Cohort Slow Fast equiv) administered Comment Part1 6 6 5 ½ Dosed on Day 1 Part 1 6 6 10 1 Dosed on Day 2 Part 1 6 6 20 2Dosed on Day 3 Part 1 2 2 30 2½ Sentinel Subjects, dosed on Day 4 Part 14 4 30 2½ Dosed on Day 7Part 2

Part 2 consisted of 14 subjects, who did not participate in Part 1,separated into 2 groups, as follows:

Part 2, Group 1: Four sentinel subjects were selected after determiningtheir acetylator status. Two were fast acetylators and two were slowacetylators. All four were dosed on Day 1 with 4 oral doses of 20 mg of3,4-DAP phosphate (given at 4-hour dosing intervals). See Table 2. OnDays 1 and 2, safety and tolerability data was collected. Subjects leftthe clinic on Day 2. The safety and tolerability data collected forthese 4 sentinel subjects were reviewed. The safety and tolerabilityresults of Days 1 and 2 for these subjects were acceptable and thus Part2 Group 2 was commenced. Minimally acceptable safety was defined as theabsence of clinical seizure activity. Subjects in Group 1 returned tothe clinic for their follow-up assessments 2-4 days following their lastdose. No seizures occurred in Part 2 Group 1.

TABLE 2 3,4-DAP Number phosphate Firdapse ® of Subjects Dose (mg baseTablets Cohort Slow Fast equiv) administered Comment Part 2, 2 2 20 2Sentinel Subjects: Group 1 Day 1 QID Dosing only Part 2, 5 5 20 2 3 DaysQID Group 2 Dosing with 4^(th) day AM single dose

Part 2, Group 2: No seizures occurred in Part 2 Group 1 and 10 subjectswere dosed on Days 1-3 with 4 oral doses of 20 mg 3,4-DAP phosphate perday (given at 4-hour dosing intervals). Each subject was given a finalmorning dose of 20 mg 3,4-DAP phosphate on Day 4. See Table 2. Subjectsin Group 2 returned to the clinic for their follow-up assessments 2-4days following their last dose.

Screening

The following assessments, procedures, and evaluations were performed aspart of the screening process: medical history, clinical laboratorytests, vital signs, physical examination, 12-lead electrocardiogram(ECG), electroencephalogram (EEG), acetylation phenotyping (1 samplefrom urine pooled over a 6-hour period), NAT2 genotyping, drugscreening, hepatitis B surface antigen (HBsAg) status, HCV status,anti-HIV 1 and 2 status, urine cotinine, and pregnancy (females ofchildbearing potential only). Drug screening, urine cotinine, pregnancytesting (females only), and clinical laboratory testing were repeatedupon admission to the clinic (Assessment Phase).

Assessments

Assessment Periods are defined as follows.

-   -   Part 1: Assessments took place in clinic from 18 hours before        study drug administration on Day 1 and up to 24 hours after last        drug administration on Day 4 for sentinel fast acetylator        subjects, on Day 5 for sentinel slow acetylator subjects, and        from 18 hours before study drug administration on Day 1 and up        to 24 hours after last drug administration on Day 7 for        non-sentinel subjects.    -   Part 2 Group 1: Assessments took place in clinic from 18 hours        before first study drug administration on Day 1 and up to 24        hours after first drug administration on Day 1.    -   Part 2 Group 2: Assessments took place in clinic from 18 hours        before first study drug administration on Day 1 and up to 24        hours after drug administration on Day 4.

Urine Collection for Phenotyping: For acetylation phenotyping, urinesamples were collected for 6 hours after a single oral dose of caffeineon Day −7. A single sample from the pooled volume was analyzed. SeeExample 15a.

Blood Sampling for NAT2 Genotyping and PK: For all subjects, a bloodsample for NAT2 genotyping was collected on Day −1. Additional bloodsampling was conducted during the assessment period for Part 1 and Part2 of the study, as follows:

Part 1 (single daily dosing): To determine the PK of amifampridine and3-N-acetyl amifampridine in plasma following single daily doses of 5,10, 20 and 30 mg, blood samples were taken pre-dose (between waking upand dosing) and at 10, 20, 30, 45, 60, 75 min, and 1.5, 2, 4, 6, 8, 10,12, 16 and 24 hours post-dose after each single daily dose [pre-dosedraws on Days 2-3 (all subjects) and Day 4 (sentinel fast acetylatorsubjects only) are the same draws as the 24-hour draw from the previousdose].

Part 2 Group 1 (all sentinel subjects): To determine the PK ofamifampridine and 3-N-acetyl amifampridine at 20 mg QID (4 times a day)for one day, blood samples were taken on Day 1 as follows:

-   -   Doses 1 and 3: pre-dose and 10, 20, 30, 45, 60, 75 min, and 1.5,        2, 3, and 4 hours post-dose    -   Doses 2 and 4: 0.5, 1, 1.5, 2, and 4 hours post-dose)    -   Dose 3 was administered following a snack and doses 1, 2, and 4        were administered following a meal.

Part 2 Group 2 (multiple daily dosing): For PK of amifampridine and3-N-acetyl amifampridine in plasma following 20 mg QID (4 times a day)oral dosing, blood samples were taken, as follows:

-   -   Day 1 and Day 3, Doses 1 and 3 on each day: pre-dose and 10, 20,        30, 45, 60, 75 min, and 1.5, 2, 3, and 4 hours post-dose; Doses        2 and 4: 0.5, 1, 1.5, 2, and 4 hours post-dose. Dose 3 was        administered following a snack; doses 1, 2, and 4 were        administered following a meal.    -   Day 2: Trough sample collected in the morning (pre-Dose 1) and        evening (pre-Dose 3)    -   Day 4: pre-dose and 10, 20, 30, 45, 60, 75 min, and 1.5, 2, 4,        6, 8, 10, 12, 16, and 24 hours post-dose.

For PK sampling 2 mL of blood was collected into the appropriate Lithiumheparin polyethylene teraphthalate collection tubes. Tubes were gentlyinverted approximately 5 times to afford mixing before processing. Afterdrawing and inverting, the blood sample was immediately transferred toan ice water bath. The samples were processed within 30 min bycentrifugation for 10 min at 4° C. and 1500×g. The plasma wastransferred into 3 separate 2 mL polypropylene tubes (2 primary samplesand 1 back-up sample) which were immediately capped. The PK plasmasamples were immediately frozen and stored at −20° C. until analysis.

Plasma concentrations of 3,4-DAP and 3-N-Acetyl 3,4-DAP metabolite weremeasured using a validated liquid chromatography and tandem massspectrometric detection (LC-MS/MS) method (Example 17b) employing anindividual stable isotope labeled internal standard for each analyte3,4-DAP and 3-N-acetyl 3,4-DAP metabolite.

Safety assessments: During the assessment period, the following safetymeasures were evaluated: vital signs, adverse events (AEs), 12-lead ECG,EEG and clinical laboratory tests.

Follow-up: Upon completion of the assessment period, the followingprocedures and evaluations were performed: adverse events, previousconcomitant medications, clinical laboratory tests, pregnancy status(females of childbearing potential only), vital signs, physicalexamination, and 12-lead ECG and EEG.

Diagnosis and all Criteria for Inclusion and Exclusion:

Individuals eligible to participate in this study had the ability andwillingness to abstain from alcohol, methylxanthine-containing beveragesor food (e.g., coffee, tea, cola, chocolate, “power drinks”), poppy seedand grapefruit (juice) 48 hours prior to admission and during clinicstays.

Individuals who met any of the following exclusion criteria were noteligible to participate in the study: subjects with, or with a recenthistory of, any clinically significant neurological, gastrointestinal,renal, hepatic, cardiovascular, psychiatric, respiratory, metabolic,endocrine, hematological or other major disorders; subjects with a priorhistory of seizures; and for Part 2 only: subjects with an NAT2 activity(ratio 1, Example 15a) below 0.06.

Investigational Product, Dose, Route and Regimen

IP was provided as scored tablets containing the equivalent of 10 mgamifampridine free base, which was administered orally. Doses persubject arc as described below based on the content of activeamifampridine ingredient. All doses were given within 30 min followingingestion of a standard meal or snack.

Part 1 Single-dose Study (12 Subjects): Each of 12 subjects received 4ascending single doses of 3,4-DAP phosphate in a sequential manner (5mg, 10 mg, 20 mg, and 30 mg). All 12 subjects were administered thefirst 3 doses (i.e., 5-20 mg) on 3 consecutive days (Days 1-3). Two fastand 2 slow acetylators were selected as the first 4 enrolled subjects.For the 2 fast acetylators, the 30 mg dose was administered on Day 4. Noseizures were seen within 24 hours of dosing, and the remaining 2 slowacetylators were dosed on Day 5. No seizures were seen in the twoacetylators dosed on Day 5. Safety and tolerability data collected forthese 4 subjects were reviewed. The safety and tolerability results ofthe 30 mg dose for these 4 sentinel subjects were acceptable, and the 30mg dose was given to the remaining 8 non-sentinel subjects on Day 7.Sentinel and non-sentinel subjects were released from the clinic on Day5, Day 6, and Day 8 respectively, with all subjects returning forfollow-up assessments 2-4 days following their last dose.

Part 2 Group 1 (4 Subjects): Four subjects received 4 single doses of 20mg 3,4-DAP phosphate given 4 hours apart on 1 day only.

Part 2 Group 2 (10 Subjects): No seizures occurred in Part 2 Group 1,and 10 subjects received drug as follows: on days 1-3, 4 single dosesper day of 20 mg 3,4-DAP phosphate given 4 hours apart and on day 4, 1dose at 20 mg 3,4-DAP phosphate.

Duration of Treatment

Subjects in Part 1 were dosed for 4 days. Subjects in Part 1 wereadministered 4 single ascending doses of 3,4-DAP phosphate (5 mg, 10 mg,20 mg, and 30 mg). For the 2 sentinel fast acetylator subjects, dosingwas completed on Days 1-4; for the 2 sentinel slow acetylator subjects,dosing was completed on Days 1-3 and on Day 5; for 8 non-sentinelsubjects, dosing was completed on Days 1-3 and on Day 7.

Part 2 subjects were dosed for 1 or 4 days. Part 2 Group 1 subjects wereadministered 4 doses of 20 mg 3,4-DAP phosphate on Day 1 only. Part 2Group 2 subjects were administered 4 doses of 20 mg 3,4-DAP phosphate oneach of Days 1-3 and a single terminal morning dose on Day 4.

Criteria for Evaluation

Pharmacokinetics: Plasma concentrations of amifampridine and its3-N-acetyl metabolite were evaluated. The PK parameters calculated wereC_(max), t_(max), k_(el), t_(1/2), AUC_(0-t), AUC_(0-t), AUC_(0-inf), %AUC_(0-inf), CL/F, Vz/F, R_(ac), where C_(max), t_(max), t_(1/2), CL/F,and Vz/F are defined in the Abbreviations table and the others aredefined as follows:

k_(el) = elimination rate constant AUC_(0-last) = area under the plasmaconcentration-time curve up to time t, where t is the last time pointwith concentrations above the lower limit of quantitation AUC_(0-inf) =total AUC after extrapolation from time t to time infinity, where t isthe last time point with a concentration above the lower limit ofquantitation AUC_(0-inf) = AUC_(0-last) + Ct/k_(el), where Ct is thelast measurable plasma concentration % AUC = percentage of estimatedpart for the calculation of AUC_(0-inf) : ((AUC_(0-inf) −AUC_(0-t))/AUC_(0-inf)) * 100% AUC_(0-τ) = area under the plasmaconcentration time curve over a dosing interval τ R_(ac) = accumulationratio, based on AUC_(0-τ) of Day 4 versus Day 1For exploratory purposes, the PK of amifampridine and 3-N-acetylamifampridine were assessed with urine caffeine acetylation activity andNAT2 enzyme allelic genotyping results.

Safety: Safety was assessed by the incidence of AEs, as well as changesin vital signs, 12-lead ECG, EEG, and clinical laboratory evaluations.

Efficacy: No efficacy analyses were performed for this study.

Statistical Methods

Pharmacokinetics: All subjects who were administered at least 1 dose ofstudy drug during each part of the study were included in the PKevaluation of that part. Dose proportionality analysis and descriptivestatistics were performed using available PK data collected during Part1; descriptive statistics were performed using available PK datacollected during Part 2.

Safety: All subjects who received any amount of study drug in eitherPart 1 or Part 2 of this study and had post-dose safety information wereincluded in the safety analyses. Safety was evaluated separately forPart 1 and Part 2 and was summarized by dose levels where appropriate.All verbatim AE terms were coded using Medical Dictionary for RegulatoryActivities (MedDRA) terminology. Only treatment-emergent AEs (i.e., AEswith a start date on or after the first dose of study drug) weresummarized. The incidence of AEs and SAEs were summarized for each studypart by dose level, system, organ, class, preferred term, relationshipto study drug, and severity. No deaths during the study, nodiscontinuations of study drug, and no withdrawals from the study andstudy drug due to an AE were observed.

Pharmacokinetics Results

Part 1

FIG. 24a gives a summary of the mean pharmacokinetic parameters of3,4-DAP in slow and fast acetylator phentoypes and FIG. 24b summarizesdata for the metabolite. After single oral doses of Firdapse® rangingfrom 5 to 30 mg, a mean C_(max) of 3.98-25.5 ng/mL was reached at0.75-1.04 hours post-dose in the fast acetylator phenotype and a meanC_(max) of 17.9-89.6 ng/mL was reached in 0.83-1.29 hours post-dose inslow acetylator phenotypes. Across each of the single dose groups, theratio of mean C_(max), slow acetylators to C_(max), fast acetylatorsranged from 3.5 to 4.5 fold (FIG. 22 and FIG. 24a ). Mean plasmaconcentration-time profiles of 3,4-DAP phosphate for single-dose cohortsin the fast and slow acetylator groups are presented in FIGS. 25a, 25b,25c, and 25d . Mean terminal elimination half-lives were between 0.603and 1.65 hours in fast acetylators and between 2.22 and 3.11 hours inslow acetylator phenotypes (FIG. 24a ). The C_(max) values increasedessentially in a dose proportional manner with respect to a 6-fold doserange (5 to 30 mg) in both the slow (5.01 fold increase) and fast (6.41fold increase) acetylator groups (FIG. 24a ). The AUC values increasedin a greater than dose proportional manner with respect to a 6-fold doserange (5 to 30 mg) in both the slow (7.64 fold increase) and fast (15.1fold increase) acetylator groups (FIG. 23 and FIG. 24a ). There werestatistically significant differences (Students t-test: P≤0.01) inexposure and elimination PK parameters C_(max), AUC, CL/F (observedclearance) and elimination t % A between the fast and slow acetylatorgroups at all dose levels (FIG. 24a ).

Differences in PK parameters between the two phenotypes is summarized byratios of C_(max), AUC, t½ and observed clearance (CL/F) for 3,4-DAPphosphate in Table 3. Over the 5-30 mg single doses the BMN125 ratios ofslow/fast ranged 3.5 to 4.50 fold for C_(max), 5.29 to 10.4 fold forAUC, 1.88 to 3.68 for t½, and 0.114 to 0.196 for observed clearance.Similarly for the metabolite the ratios of slow/fast ranged 0.498 to0.540 for C_(max), 0.661 to 0.717 for AUC, 1.13 to 1.22 for t½ and 1.41to 1.51 for observed clearance (FIG. 24b ).

TABLE 3 For Cohort 1, Mean Ratios for 3,4-DAP phosphate Single Dose PKParameters for Slow vs. Fast Acetylator Phenotypes: C_(max), AUC,Elimination T ½ and Clearance Mean Dose Mean C_(max) AUC_(0-t) Meant_(1/2) Mean CL/F Level Slow/fast Slow/fast Slow/fast Slow/fast Ratio(mg) Ratio^(a) Ratio^(a) Ratio^(a) Ratio^(a) 3,4-DAP 5 4.50 10.4 3.680.114 10 3.47 6.94 2.15 0.163 20 3.50 5.75 2.38 0.186 30 3.51 5.29 1.880.196 ^(a)Figures are ratios of mean parameter values of Slow/Fastacetylator phenotypes

3,4-DAP data for Part 1 is summarized in Table 7 (day 1 of multipledosing, fast and slow), Table 8 (day 3 of multiple dosing, fast andslow), Table 9 (day 4 of multiple dosing, fast and slow), and for themetabolite in Table 10 (day 1 of multiple dosing, fast and slow), Table11 (day 3 of multiple dosing, fast and slow), and Table 12 (day 4 ofmultiple dosing, fast and slow). In Part 1 of the study there werestatistically significant differences in PK parameters for exposure andelimination between the fast and slow acetylator groups at all doselevels indicate that metabolic clearance of Firdapse® by N-acetylationsignificantly impacts the plasma pharmacokinetic profile of oralFirdapse®.

Part 2

Mean plasma concentration-time profiles for multiple-dose cohorts in thefast and slow acetylator groups are presented graphically in FIGS. 26a,26b, and 26c (dosing days 1, 3 and 4, respectively). Mean plasmaconcentration-time plots of 3-N-acetyl metabolite for multiple-dosecohorts in the fast and slow acetylator groups are presented graphicallyin FIGS. 27a, 27b, and 27c (days 1, 3 and 4, respectively).

In the multiple-dose part 2 study (20 mg Firdapse®, QID, Q4 hr),Firdapse® plasma concentrations increased and declined within each ofthe dosing intervals evaluated on PK sampling days 1, 3 and 4 indicatingrapid absorption, distribution and plasma clearance of Firdapse® withfast acetylator phenotypes consistently having the lowest 3,4-DAP plasmaconcentrations (FIGS. 26a, 26b, and 26c ), and the highest metaboliteplasma concentrations (FIGS. 27a, 27b, and 27c ). On Days 1-4 ofFirdapse® dosing, 3,4-DAP plasma mean C_(max) values ranged from13.3-24.4 ng/mL in fast acetylators and from 67.1-97.1 ng/mL in slowacetylators (Table 7, 8, and 9). Firdapse® exposure measured by AUCindicated a range of dose interval AUC_(0-4h) values of 22.5-28.5ng-h/mL in fast acetylators and 115-168 ng-h/mL in slow acetylators onDays 1-4 of dosing (Table 7, 8, and 9). The terminal elimination halflife for Firdapse® on dose day 4, after 3 days of subsequent QID dosing,was calculated to be 1.95 h in fast and 3.24 h in the slow acetylatorgroup (Table 7, 8, and 9). Apparent volume of distribution (Vdz) andobserved clearance (CL/F) were categorized as high (Vz>350 L, CL/F>85L/h; 70 kg human) in both phenotypes for Firdapse® (Table 7, 8, and 9).Corresponding data is given for N-(4-aminopyridin-3-yl)acetamide inTables 10, 11, and 12.

TABLE 7 Mean PK Parameter Summary for 3,4-DAP for Fast and SlowAcetylators on Day 1 of Multiple Dosing (20 mg, QID) AUC₀₋₄ AUC₀₋₂₄C_(max) T_(max) t_(1/2) Vz/F CL/F Dose Int. (ng · h/mL) (ng · h/mL)(ng/mL) (hr) (hr) (L) (L/hr) Mean PK Parameters Day 1 for FastAcetylators (Phenotype) Dose 1 Arith. Mean 23.5 24.4 0.809 1.12 1334 898SD 9.34 17.6 0.412 0.272 386 421 CV (%) 39.7 72.2 50.9 24.4 28.9 46.9Dose 2 Arith. Mean 22.5 13.3 1.07 1.31 1336 757 SD 9.81 7.58 0.450 0.419210 225 CV (%) 43.6 57.2 42.0 32.0 15.7 29.7 Dose 3 Arith. Mean 24.116.8 0.583 1.30 1725 879 SD 11.1 6.59 0.168 0.250 1132 466 CV (%) 45.939.3 28.7 19.2 65.6 53.0 Dose 4 Arith. Mean 23.7 16.4 0.643 1.49 1732885 SD 8.61 5.76 0.244 0.590 847 531 CV (%) 36.4 35.1 38.0 39.7 48.960.0 Dose 1-4 Arith. Mean 97.9 SD 39.7 CV (%) 40.6 Mean PK ParametersDay 1 for Slow Acetylators (Phenotype) Dose 1 Arith. Mean 115 74.8 1.121.39 299 152 SD 22.4 36.8 0.536 0.257 48.4 29.7 CV (%) 19.6 49.1 47.918.5 16.2 19.5 Dose 2 Arith. Mean 152 83.7 1.14 1.93 293 107 SD 17.935.1 0.690 0.443 60.1 17.3 CV (%) 11.8 42.0 60.4 22.9 20.5 16.3 Dose 3Arith. Mean 140 72.2 0.857 1.59 269 117 SD 25.9 17.3 0.197 0.168 52.529.3 CV (%) 18.5 24.0 22.9 10.6 19.5 25.1 Dose 4 Arith. Mean 145 67.11.29 2.52 338 101 SD 35.6 20.9 1.32 0.935 73.7 35.3 CV (%) 24.6 31.1 10337.1 21.8 34.9 Dose 1-4 Arith. Mean 630 SD 112 CV (%) 17.8

TABLE 8 PK Parameter Summary for 3,4-DAP for Fast and Slow Acetylatorson Day 3 of Multiple Dosing (20 mg, QID) AUC₀₋₄ AUC₀₋₂₄ C_(max) T_(max)t_(1/2) Vz/F CL/F Dose Int. (ng · h/mL) (ng · h/mL) (ng/mL) (hr) (hr)(L) (L/hr) Mean PK Parameters Day 3 for Fast Acetylators (Phenotype)Dose 1 Arith. Mean 24.9 24.0 0.800 1.07 1387 973 SD 12.2 18.1 0.2740.209 753 695 CV (%) 49.1 75.6 34.2 19.6 54.3 71.5 Dose 2 Arith. Mean28.5 18.7 0.900 1.34 1112 607 SD 17.3 10.9 0.652 0.258 310 248 CV (%)60.9 58.5 72.4 19.2 27.8 40.8 Dose 3 Arith. Mean 25.8 21.9 0.532 1.732200 817 SD 13.8 12.4 0.211 0.573 1753 404 CV (%) 53.5 56.6 39.6 33.179.7 49.4 Dose 4 Arith. Mean 25.1 16.2 0.800 1.82 1867 795 SD 13.2 5.610.274 0.638 701 413 CV (%) 52.7 34.6 34.2 35.1 37.5 51.9 Dose 1-4 Arith.Mean 111 SD 62.1 CV (%) 55.9 Mean PK Parameters Day 3 for SlowAcetylators (Phenotype) Dose 1 Arith. Mean 130 81.2 1.00 1.60 283 124 SD28.8 51.9 0.395 0.239 47.9 19.3 CV (%) 22.3 64.0 39.5 14.9 16.9 15.6Dose 2 Arith. Mean 164 96.1 1.00 1.97 260 92.9 SD 17.7 35.0 0.612 0.69682.6 8.19 CV (%) 10.8 36.4 61.2 35.4 31.8 8.81 Dose 3 Arith. Mean 15680.9 0.800 1.63 241 103 SD 21.7 20.4 0.274 0.177 34.0 12.5 CV (%) 13.925.2 34.2 10.9 14.1 12.1 Dose 4 Arith. Mean 168 97.1 0.600 3.56 376 73.2SD 21.6 34.2 0.224 0.492 75.7 10.0 CV (%) 12.9 35.3 37.3 13.8 20.1 13.7Dose 1-4 Arith. Mean 701 SD 74.3 CV (%) 10.6

TABLE 9 Mean PK Parameter Summary for 3,4-DAP for Fast and SlowAcetylators (Phenotype) on Day 4 of Multiple Dosing (20 mg, Single Dose)AUC₀₋₄ AUC₀₋₂₄ C_(max) T_(max) t_(1/2) Vz/F CL/F (ng · h/mL) (ng · h/mL)(ng/mL) (hr) (hr) (L) (L/hr) Mean PK Parameters Day 4, Single Dose FastArith. Mean 22.6 25.9 13.6 0.900 1.95 1774 673 SD 10.0 12.9 6.60 0.4540.723 388 195 CV (%) 44.1 49.9 48.4 50.5 37.0 21.9 29.0 Slow Arith. Mean133 186 72.5 1.20 3.24 486 108 SD 21.9 32.5 43.9 0.597 1.03 91.3 17.6 CV(%) 16.5 17.5 60.6 49.7 31.9 18.8 16.3

TABLE 10 Mean PK Parameter Summary for 3-N-Acetyl Metabolite in Fast andSlow Acetylators on Day 1 of Multiple Dosing (20 mg, QID) AUC₀₋₄ AUC₀₋₂₄C_(max) T_(max) t_(1/2) Vz/F CL/F Dose Int. (ng · h/mL) (ng · h/mL)(ng/mL) (hr) (hr) (L) (L/hr) Mean PK Parameters Day 1 for FastAcetylators (Phenotype) Dose 1 Arith. Mean 773 333 1.43 2.35 55.2 16.2SD 111 79.7 0.875 0.191 11.6 2.32 CV (%) 14.3 23.9 61.2 8.15 21.0 14.3Dose 2 Arith. Mean 1160 396 1.50 2.93 32.9 7.78 SD 236 92.6 0.289 NC NCNC CV (%) 20.4 23.4 19.2 NC NC NC Dose 3 Arith. Mean 1277 426 0.964 3.2840.3 8.90 SD 253 76.9 0.336 0.818 7.10 2.23 CV (%) 19.8 18.0 34.9 24.917.6 25.0 Dose 4 Arith. Mean 1341 433 1.14 3.93 40.0 7.62 SD 272 73.40.378 1.08 4.64 2.53 CV (%) 20.3 16.9 33.1 27.5 11.6 33.2 Dose 1-4Arith. Mean 5744 SD 1221 CV (%) 21.3 Mean PK Parameters Day 1 for SlowAcetylators (Phenotype) Dose 1 Arith. Mean 383 150 1.68 2.95 120 28.8 SD62.4 21.5 0.657 0.405 14.5 6.34 CV (%) 16.3 14.3 39.1 13.8 12.1 22.0Dose 2 Arith. Mean 627 204 1.46 3.50 101 20.2 SD 118 22.6 0.588 0.6573.17 3.17 CV (%) 18.9 11.1 40.4 18.8 3.15 15.7 Dose 3 Arith. Mean 759240 1.25 4.00 71.5 13.5 SD 185 53.7 0.289 1.19 14.3 5.58 CV (%) 24.422.3 23.1 29.9 20.0 41.3 Dose 4 Arith. Mean 769 235 1.71 4.74 70.6 10.4SD 200 61.8 1.07 1.04 16.1 1.57 CV (%) 26.0 26.3 62.7 22.0 22.8 15.1Dose 1-4 Arith. Mean 3576 SD 623 CV (%) 17.4

TABLE 11 Mean PK Parameter Summary for 3-N-Acetyl Metabolite in Fast andSlow Acetylators on Day 3 of Multiple Dosing (20 mg, QID) AUC₀₋₄ AUC₀₋₂₄C_(max) T_(max) t_(1/2) Vz/F CL/F Dose Int. (ng · h/mL) (ng · h/mL)(ng/mL) (hr) (hr) (L) (L/hr) Mean PK Parameters Day 3 Phenotype: FastAcetylators Dose 1 Arith. Mean 1019 374 1.35 2.77 44.5 11.5 SD 201 60.50.224 0.511 4.55 2.81 CV (%) 19.7 16.2 16.6 18.5 10.2 24.4 Dose 2 Arith.Mean 1319 448 1.30 3.60 36.6 7.56 SD 281 98.6 0.447 0.94 4.12 2.98 CV(%) 21.3 22.0 34.4 26.0 11.3 39.3 Dose 3 Arith. Mean 1413 490 0.900 3.7439.2 7.72 SD 347 127 0.137 0.945 6.67 2.54 CV (%) 24.6 26.0 15.2 25.217.0 32.9 Dose 4 Arith. Mean 1456 509 1.10 4.17 42.0 7.03 SD 285 1010.652 0.611 6.01 1.02 CV (%) 19.6 19.9 59.3 14.6 14.3 14.5 Dose 1-4Arith. Mean AUC0-24 6567 SD 1569 CV (%) 23.9 Mean PK Parameters Day 3Phenotype: Slow Acetylators Dose 1 Arith. Mean 523 206 1.75 3.47 95.019.0 SD 65.9 67.1 0.901 0.437 11.4 0.118 CV (%) 12.6 32.5 51.5 12.6 12.00.620 Dose 2 Arith. Mean 684 222 1.50 3.89 81.5 14.5 SD 81.6 29.0 0.5000.055 2.60 0.672 CV (%) 11.9 13.1 33.3 1.43 3.19 4.62 Dose 3 Arith. Mean802 249 1.20 3.75 68.1 12.7 SD 187 59.8 0.274 0.672 14.7 2.75 CV (%)23.4 24.0 22.8 17.9 21.7 21.6 Dose 4 Arith. Mean 814 258 0.900 5.70 78.39.65 SD 134 25.0 0.224 0.643 13.7 2.15 CV (%) 16.5 9.72 24.8 11.3 17.522.3 Dose 1-4 Arith. Mean 3649 SD 654 CV (%) 17.9

TABLE 12 Mean PK Parameter Summary for 3-N-Acetyl Metabolite Fast andSlow Acetylators on Day 4 of Multiple Dosing (20 mg, Single Dose) AUC₀₋₄AUC₀₋₂₄ C_(max) T_(max) t_(1/2) Vz/F CL/F (ng · h/mL) (ng · h/mL)(ng/mL) (hr) (hr) (L) (L/hr) Mean PK Parameters Day 4, Single Dose FastArith. Mean 1028 2125 2206 363 1.40 4.95 69.4 SD 213.7 756 835 53.40.379 0.988 20.2 CV (%) 20.8 35.6 37.8 14.7 27.1 20.0 29.2 Slow Arith.Mean 513 1229 1278 178 1.55 4.99 116 SD 74.4 249 265 34.7 0.512 0.64222.4 CV (%) 14.5 20.3 20.7 19.5 33.1 12.9 19.4

The differences in PK parameters or exposure between the last and slowacetylator groups at all single and multiple dose levels indicateFirdapse PK parameters are significantly influenced by NAT phenotype andthat metabolic clearance of Firdapse by N-acetylation significantlyimpacts the plasma pharmacokinetic profile of orally administeredFirdapse® tablets.

Safety Results

There were no serious adverse events. There were no clinicallysignificant changes in the subjects' ECG and there were no otherclinically significant changes observed in other clinical laboratoryassessments. No subject discontinued due to any adverse event. Alltreatment-emergent adverse events were mild and transient and resolvedwithout sequela.

Part 1: Treatment-emergent drug-related adverse events by treatment andphenotype are given in FIG. 28. All adverse events were observed in slowacetylators only, even at the higher doses of 20 mg (Day 3) and 30 mg(Days 4-7).

Part 2: Treatment-emergent drug-related adverse events by treatment andphenotype are given in FIG. 29. Adverse events were observed in bothacetylator types. Forty-five drug-related adverse events were reportedby 6 (86%) of 7 slow acetylators. In contrast, six drug-related adverseevents were reported in 3 (43%) of 7 fast acetylators.

Example 19 Clinical Trial to Evaluate Efficacy, Safety, and RelationshipBetween NAT Genetic Polymorphism Status and Plasma Levels ofAmifampridine and 3-N-Acetyl Amifampridine in LEMS Patients

Efficacy: In this study efficacy of amifampridine phosphate versusplacebo on muscle strength in patients with LEMS at the end of a 14-daydiscontinuation period is to be evaluated. In addition, efficacy can bedetermined by determining walking speed in patients with LEMS at the endof a 14-day discontinuation period. The following parameters in patientswith LEMS at the end of a 14-day discontinuation period can also bedetermined in evaluating efficacy: CMAP: amplitude, CGI-S:Investigator-perceived improvement in illness severity, CGI-I:Investigator-perceived global improvement or change, and SGI: Subjectglobal impression of improvement. Finally, this study is designed to 1)confirm the exposure of amifampridine and its major metabolite,3-N-acetyl amifampridine, based on plasma concentrations in the LEMSpatient population; 2) to evaluate the relationship between NAT geneticpolymorphism status and plasma levels of amifampridine and 3-N-acetylamifampridine in LEMS patients.

Safety: The safety objective of the study is to assess the safety,including the long-term safety, of amifampridine phosphate in patientswith LEMS.

Study Design

This double-blind, placebo-controlled, randomized (1:1) discontinuationstudy is a 4-part study designed to evaluate the efficacy and safety ofmultiple dose administration of amifampridine phosphate in patients withLEMS. The long-term clinical efficacy of amifampridine phosphate isevaluated in patients with at least 91 days of previous amifampridinetreatment by comparing changes (as determined by QMG) that occur inpatients who discontinue treatment versus patients who continue onactive treatment to the end of a 14-day double-blind efficacy evaluationperiod.

In addition to amifampridine, patients will continue to receive bestsupportive care (BSC) treatment as determined by the investigator usingconcomitant medications permitted per the protocol, which include thefollowing: 1) selected oral immunosuppressants (e.g., prednisone orother corticosteroids, azathioprine, mycophenolate) and 2) peripherallyacting cholinesterase inhibitors (e.g., pyridostigmine). No BSC changesshould be made during Screening, Part 1 (Open-label Run-in), Part 2(Double-blind Treatment Discontinuation), and Part 3 (Double-blindTreatment). However, modifications is allowed in Part 4 (Open-labelExtension). If changes are made to BSC during Screening or Part 1, entryof the patient into Part 2 may be delayed to allow stabilization of thenew regimen.

An analysis for both efficacy and safety is performed after all patientscomplete Part 3, Double-blind Treatment. A final safety analysis isperformed at the end of the study, completion of Part 4, Open-labelExtension.

Screening and Enrollment

After providing informed consent, patients undergo a Screeningevaluation to determine study eligibility. Efficacy assessments isperformed for all patients during Screening. For patients receivingamifampridine treatment prior to enrollment, efficacy assessments isperformed at standardized times relative to the first dosing time of theday (see the standardized schedule of efficacy assessments below). Forpatients not receiving amifampridine treatment at the time of Screening,efficacy assessments should be completed on the same standardizedschedule starting at a time consistent with anticipated future firstdosing time of the day. Safety assessments are also be conducted duringScreening.

Part 1: Open-Label Run-In

The purpose of the Open-label Run-in is to 1) allow the investigator totitrate amifampridine phosphate to the optimal dose regimen for eachindividual patient, and 2) to allow patients to achieve duration ofamifampridine exposure required prior to randomized discontinuation.

All patients will start taking amifampridine phosphate on Day 1 of theOpen-label Run-in. The dose of amifampridine is individually determined,for example, by the investigator, within the bounds of a 30-80 mg totaldaily dose and a maximum of 20 mg at any single administration. Patientswith moderate renal impairment will start at a total dose of 10 mg perday.

Investigational Product(s), Dose, Route, and Regimen.

The final amifampridine phosphate dose regimen during Open-Label Run-inis determined by the investigator for each individual patient, based onthe optimal observed neuromuscular benefit. Patients who do not achieveoptimal neuromuscular benefit during this time period will bediscontinued from treatment and withdrawn from the study followingsafety follow-up.

Patients are required to have a minimum of 91 days (13 consecutiveweeks) of amifampridine, base or phosphate, treatment prior torandomization into Part 2, Double-blind Treatment Discontinuation. The91 consecutive days may include time receiving amifampridine prior toScreening, during Screening, and during Run-in. Patients are alsorequired to have a stable amifampridine phosphate dose regimen (i.e.,the same total daily dose and dose regimen) for at least the 7 daysimmediately prior to entering Part 2. If at least 1 day with more than50% of doses is missed during the 7 days immediately prior to enteringPart 2, the 7 day run-in period must be restarted. If a patient requiresmore than 2 restarts, the investigator should contact the MedicalMonitor to discuss.

Patients should remain on the same BSC therapy throughout the Run-inperiod. If a change in concomitant immunotherapy is made, the required91 days of consecutive treatment is restarted at that point. If a changein peripheral cholinesterase inhibitor is made during the 7 days of astable amifampridine phosphate regimen, then that 7-day period isrestarted at that point. Thus changes in BSC can delay patient entryinto Part 2.

Optional blood samples for NAT genetic testing will be collected on Day1 of the Open-Label Run-in. Safety assessments will be performedbi-weekly per the schedule of assessments and as necessary.Electrocardiogram (ECG) will be performed on Day 1 and then monthly perthe schedule of assessments.

Patients should begin Part 2 (Double-blind Treatment Discontinuation.Randomization) as soon as possible after meeting all of the Open-labelRun-in requirements described above: 1) optimal neuromuscular benefit onamifampridine phosphate treatment; 2) at least 91 consecutive days ofamifampridine treatment (base or phosphate); 3) at least 7 consecutivedays of stable open-label amifampridine phosphate dosing (i.e., the sametotal daily dose and dose regimen) immediately prior to entering Part 2;and 4) required duration of a stable BSC regimen.

Part 2: Double-Blind Treatment Discontinuation

On Day 1 of Part 2, patients are randomized (1:1) using a centralizedrandomization method (interactive voice/web response system, IXRS) toone of the following blinded treatments:

-   -   Continuation of Treatment (treatment group A): Amifampridine        phosphate (at a dose established during Open-label Run-in) for 7        days.    -   Discontinuation of Treatment (treatment group B): This involves        downward titration of amifampridine phosphate dose to 0 mg. This        will be accomplished by substituting an increasing proportion of        matching placebo tablets for amifampridine phosphate tablets        starting on Day 2 and ending on Day 7, at which point all        tablets are placebo.

Blood samples are collected for pharmacokinetic analysis on Day 1 ofPart 2 prior to dosing, and at 2.0 and 4.0 hours (both±15 min) afterdosing. Blood samples for PK are collected on Day 2 of Part 2 prior todosing, at 15, 30 and 90 min (all±5 min) post-dosing, and at 2.0 and 4.0hours (both±15 min) post dosing. Urine samples collected on Day 1 areassayed for 3-N-acetyl amifampridine metabolite. Also on Day 2 of Part2, ECGs are obtained in triplicate prior to dosing and at 1.0 and 2.0hours (±15 min) after dosing.

Standardized Schedule of Efficacy Assessments

Baseline efficacy assessments is performed on Day 1. The first dose mustbe taken at the same time each day of Part 2 (±15 min) and administeredin the clinic on days that efficacy assessments are performed. Therequired schedule of efficacy assessments is listed in the table below.

Start Time After Dose Assessment (±15 min unless otherwise specified)CMAP amplitude 00:45 (−5/+15 min) QMG 01:15 T25FW 02:15 CGI-S, CGI-I, Inthis order following prior assessments SGITo prevent unblinding, the same individual rater may not perform boththe CMAP and QMG tests on an individual patient.Part 3: Double-Blind Treatment

Patients who are randomized to amifampridine phosphate in Part 2 willcontinue to receive the same dose regimen for 7 additional days.Patients who are downward titrated to placebo in Part 2 will remain onplacebo for 7 days.

Efficacy and safety assessments are performed on Day 8 and Day 14following the standardized schedule. Urine samples collected on Days 8and 14 are assayed for 3-N-acetyl amifampridine metabolite to verifycompliance with the prohibition against the use of other sources ofamifampridine during the Double-blind Treatment period.

Rescue Treatment

In Parts 2 and 3 of the study, it is recognized that some patients mayexperience severe signs and symptoms of their disease upon downwardtitration and cessation of amifampridine phosphate. Therefore, rescuetreatment will be provided to patients who experience treatment failureas defined by meeting at least 1 of the following criteria:

-   -   Becomes non-ambulatory (after having been ambulatory at        Screening) as defined as a patient who was previously able to        walk but who becomes unable to walk even with the use of        assistive devices (e.g., cane or walker) and requires a        wheelchair for mobility.    -   Demonstrates an increase (worsening) in QMG score by >5 points.    -   Develops respiratory failure as defined as the need for        mechanical ventilation.

Unless the clinical condition of the patient is so severe as to dictateotherwise, patients who meet rescue treatment criteria will bediscontinued from Part 2 or 3 and will proceed to Rescue Visit 1. RescueVisit 1 should be performed as soon as a patient is identified aspotentially requiring open-label amifampridine phosphate rescuetreatment. If possible, a confirmatory Rescue Visit 2 should beperformed approximately 8 to 24 hours following Rescue Visit 1. Ifpossible, efficacy and safety assessments will be performed and a venousblood sample for the determination of plasma amifampridine concentrationwill be obtained at each rescue visit. During the interval betweenRescue Visits, patients will receive standard of care as deemedappropriate by the investigator.

After completion of the assessments/procedures for Rescue Visit 2,rescue treatment may be initiated. Rescue treatment may includeopen-label amifampridine phosphate at a dose level determined by theinvestigator. Other treatments for LEMS may also be administered asjudged clinically appropriate by the investigator; however,immunosuppressives that lower the seizure threshold (e.g., cyclosporine,tacrolimus) or other aminopyridines are not permitted in combinationwith amifampridine phosphate. The patient will be provided the option toreceive open-label amifampridine phosphate in Part 4, Open-labelExtension.

Part 4: Open-Label Extension

The following patients will be offered the option to participate in Part4 Open-label Extension: 1) patients who complete Parts 1, 2, and 3; 2)patients who received per protocol rescue treatment with amifampridinephosphate; or 3) patients who are participating in Part 1 but who havenot established 7 days of stable amifampridine phosphate dosing when the30th patient is randomized in Part 2.

Amifampridine phosphate dosing is started on Day 15. Patients are seenon Day 19 (±2 days) and monthly for the next 2 visits and then quarterlythereafter. Safety assessments are performed at each visit. Patientsreceive their individualized dose of open-label amifampridine phosphatebased on investigator assessment. Investigators determine whether or notto follow dose initiation and titration as in Part 1 Open-label Run-in.Changes to BSC may be made at the investigator's discretion, as long asprohibited medications are not used. Each patient's study participationmay continue until the study is terminated (2 years after the lastpatient is enrolled into Part 4). If amifampridine phosphate is orbecomes commercially available while the patient is enrolled in Parts1-3 of the study, they may only participate in Part 4 for a maximum of 1year.

Diagnosis and all Criteria for Inclusion and Exclusion:

Individuals eligible to participate in this study must have a confirmeddiagnosis of LEMS as documented by acquired (typical) proximal muscleweakness and at least 1 of nerve conduction findings (CMAP thatincreases at least 2-fold after maximum voluntary contraction of thetested muscle) and positive anti-P/Q type voltage-gated calcium-channelantibody test; have completed an anti-cancer treatment at least 3 months(90 days) prior to Screening; have a QMG score of ≥5 is required forpatients without any prior symptomatic treatment for LEMS; present withsome signs and/or symptoms consistent with LEMS if currently receivingtreatment for LEMS; have normal respiratory function as defined by aforced vital capacity>80% predicted (score of 0 on this dimension ofQMG); and have normal swallowing function as defined by the ability toswallow 4 ounces of water without coughing or throat clearing (score of0 on this dimension of QMG).

Individuals who meet any of the following exclusion criteria are noteligible to participate in the study: history of epilepsy or seizure(including single 1-time seizure, but excluding generalized febrileseizures occurring before the age of 5 years); known active brainmetastasis; use of 4-aminopyridine and any form of 3,4-diaminopyridine;use of medications known to lower the epileptic threshold within thelonger of 7 days or 5 half-lives prior to Screening; use of selectedantidepressants of the selective serotonin uptake inhibitor (SSRI)class; use of medications which inhibit neuromuscular junction functionwithin the longer of 7 days or 5 half-lives prior to Screening; use ofguanidine hydrochloride within 7 days of Screening; use of rituximabwithin 12 months prior to Screening; treatment with a concomitantmedication that prolongs the QT/QTc interval within the longer of 7 daysor 5 half-lives prior to Screening; treatment with sultopride(4-amino-N-[(1-ethylpyrrolidin-2-yl)methyl]-5-ethylsulfonyl-2-methoxybenzamide)within 7 days prior to Screening; an electrocardiogram (ECG) atScreening that, in the opinion of the external reviewing cardiologist,shows any of sinus arrhythmia with unacceptable rate variation,excessive heart rate variation at rest, QTcB interval>450 msec, PRinterval>210 msec, QRS interval>120 msec if 35 years of age or younger,or >110 msec if over age 35 years, or early repolarization pattern thatincreases the risk of participating in the study; history ofarrhythmias, risk factors for torsade de pointes, or severe renalimpairment or evidence of severe renal impairment on Screeninglaboratory tests; likely or expected to require treatment for cancerwithin 3 months (90 days) after entering Screening; ALT, AST, and/ortotal bilirubin>ULN for patients without liver metastases; orALT/AST>5×upper limit of normal (ULN) and/or total bilirubin>3×ULN inpatients with liver metastases from cancer.

Investigational Product(S), Dose, Route, and Regimen:

IP is provided in 250 mg tablets, containing the equivalent of 10 mgamifampridine base per tablet.

During Parts 1 and 4 (open-label), patients receive a total daily doseof amifampridine phosphate 30 to 80 mg per day given 3 to 4 times perday, with a maximum single dose of 20 mg; with the exception of patientswith moderate renal impairment, who will start at a total daily dose of10 mg. Amifampridine phosphate is to be taken with food. In Part 1,determination of initial dose level are as follows:

-   -   In patients already taking amifampridine base, amifampridine        phosphate should be started at an equivalent or lower dose of        amifampridine phosphate, at the investigator's discretion.    -   In patients not currently taking amifampridine, amifampridine        phosphate should be initiated at a low dose (10 mg, 3 to 4 times        per day).    -   In patients with moderate renal impairment who are not taking        amifampridine phosphate prior to the study, the starting total        daily dose will be 10 mg. Moderate renal impairment is defined        as a creatinine clearance of 30 to 59 mL/min where creatinine        clearance is calculated using the Cockcroft-Gault equation as        [(140-Age)*Mass (in kg)]/[72*serum creatinine (in mg/dL)] (Note:        If the patient is a female, multiply the output of the equation        by 0.85.) Note, a brief deviation from the usual requirement        that the total daily dose fall in the 30-80 mg/day range is        allowed during initial titration in patients with moderate renal        impairment.        The titration schedule is an increase by 10 mg increments every        4 to 5 days to a maximum of 80 mg per day based on optimal        neuromuscular benefit and at the discretion of the investigator.

During Part 2 (Double-blind Treatment Discontinuation), patientsrandomized to treatment Group A continue for 7 days on the amifampridinephosphate dose established during the Open-label Run-in phase. Patientsrandomized to treatment Group B have their amifampridine phosphate dosedownward titrated to 0 mg. This is accomplished by substituting anincreasing proportion of matching placebo tablets for amifampridinephosphate tablets starting on Day 2 and ending on Day 7, at which pointall tablets are placebo. Patients in treatment Group B continue to takethe same number of tablets daily throughout this period.

During Part 3 (Double-blind Treatment), patients who are randomized toamifampridine phosphate in Part 2 Group A continue to receive the samedose regimen for 7 additional days. Patients who are downward titratedto placebo in Part 2 Group B remain on placebo for 7 days.

During Part 4 (Open-label Extension) patients receive theirindividualized dose of open-label amifampridine phosphate based oninvestigator assessment at 30 to 80 mg per day given 3 to 4 times perday, up to a maximum of 20 mg in a single dose.

Reference Therapy, Dose, Route, and Regimen:

The reference therapy during the Double-blind Treatment Discontinuationand Double-blind Treatment period is a placebo, provided as tabletsindistinguishable from amifampridine phosphate tablets. The placebo willbe administered consistent with the dose and dose regimen of theinvestigational product (amifampridine phosphate).

No reference therapy is to be administered in Part 1 Open-label Run-inor in Part 4 Open-label Extension.

Duration of Treatment:

Part 1: 7-91 days (Open-label Run-in); Part 2: 7 days (Double-blindTreatment Discontinuation); Part 3: 7 days (Double-blind Treatment);Part 4: Open-label Extension

To maintain the completeness and integrity of the data, patients whodiscontinue from treatment early should continue to have studyassessments performed until completion of the Double-blind Treatmentphase as long as in the judgment of the investigator such continuedparticipation would not detrimentally affect the health, safety, orwelfare of the patient.

Criteria for Evaluation:

All efficacy evaluations is obtained during the Double-Blind phase ofthe study (to Day 14).

Efficacy: Efficacy endpoints that could be evaluated include one or moreof the following the change from baseline to Day 14 in QMG score, thechange from baseline to Day 14 in walking speed assessed by the T25FWtest, change from baseline to Day 14 in CMAP amplitude, the Day 14 CGI-Iand SGI scale measurements, the proportion of patients in each treatmentgroup with a Day 14 CGI-I scale rating of 1, 2, 3, or 4, the proportionof patients in each treatment group with a Day 14 SGI scale rating of 4,5, 6, or 7, and The change from baseline to Day 14 in CGI-S scalemeasurements.

PK of amifampridine and 3-N-acetyl metabolite in plasma will beconducted in the LEMS patient population. The relationship between NATgenetic status and exposure to amifampridine and 3-N-acetyl metabolitewill be evaluated and described.

Safety: Safety will be assessed by the incidence of AEs, including SAEs.Safety will also be assessed by changes from baseline in the following:Abnormal and clinically significant laboratory tests, vital signs,physical examination, concomitant medications, and ECG.

Statistical Methods:

An analysis for both efficacy and safety is performed after all patientscomplete Part 3, Double-blind Treatment. A final safety analysis isperformed at the end of study, completion of Part 4, Open-labelExtension.

Sample Size Determination

A total of 30 patients are provided 80% power to detect a 3.0 unitdifference in the mean change of the QMG scores between the 2 treatmentgroups, assuming a type I error of 0.05 and a common standard deviationof 2.7.

Efficacy Analysis

Efficacy analysis is conducted on all patients randomized into thestudy. All endpoints arc summarized using descriptive statistics at eachmeasurement time point, including for Part 1 of the study. Treatmentdifferences will be assessed at a significance level of 0.05 for allendpoints. Baseline is defined as the measurements obtained at thebeginning of Part 2 Double-blind Treatment Discontinuation (Day 1).

The change in QMG scores from baseline (Day 1, Part 2) to Day 14 (Part3) is compared between treatment groups using an analysis of covariance(ANCOVA) where the covariate adjusted for will be baseline QMG. Ifsignificant departures from normality are observed, then the WilcoxonRank Sum test comparing the treatment groups will be performed on theQMG change from baseline scores. Alternatively a Rank ANCOVA analysismay be performed.

Change in the T25FW test walking speed from baseline to Day 14 iscompared between treatment groups, using the same analysis method whichis used for the primary efficacy endpoint.

Change in CMAP amplitude, as well as other continuous efficacyendpoints, is analyzed in a manner similar to the analysis performed forthe QMG scores and walk test. All categorical endpoints will be analyzedusing contingency table approach and the comparisons between the 2treatment groups will be performed using Fisher's Exact Test. Due to theexploratory nature of the analysis, no adjustments for multiple testingwill be done.

Pharmacokinetic and Genetic Analysis

Data from all patients who have PK blood samples taken are included inthe PK analysis to determine amifampridine and N-acetyl metaboliteexposure levels at key time points post dosing. Sparse or limitedexposure data from this study may be analyzed in combination withavailable full PK data (and PK models derived thereof) from otherclinical studies to derive exposure parameters for the LEMS patientpopulation. Amifampridine and 3-N-acetyl amifampridine exposure with NATgenotype status, along with descriptive statistics, will be evaluated.

Safety Analysis

All patients who receive at least 1 dose of IP or placebo, and have anypost-treatment safety information collected are included in the safetyanalysis. The safety analysis will be descriptive and will be performedseparately for each part of the study. All AEs are coded using theMedical Dictionary for Regulatory Activities (MedDRA). Onlytreatment-emergent AEs (TEAEs) will be included in the safety analysis.The incidence of AEs will be summarized by system organ class, preferredterm, relationship to treatment, and severity for each treatment group.All AEs, as well as AEs leading to premature discontinuation and seriousAEs (SAEs), will be listed. All other safety measures includinglaboratory tests, vital signs, ECGs, and concomitant medications datawill also be summarized descriptively.

Handling of Dropouts and Missing Values:

Missing efficacy values for the primary and secondary endpoints will beimputed. Imputation methods for missing data will be described in theStatistical Analysis Plan.

Because the completeness of the data affects the integrity and accuracyof the final study analysis, every effort will be made to ensurecomplete, accurate, and timely data collection and, therefore, avoidmissing data. Patients who discontinue from IP treatment early shouldcontinue to have study assessments performed until completion of Part 3,Double-blind Treatment, as long as in the judgment of the investigatorsuch continued participation would not detrimentally affect the health,safety, or welfare of the patient.

All patents, publications and references cited herein are hereby fullyincorporated by reference. In case of conflict between the presentdisclosure and incorporated patents, publications and references, thepresent disclosure will control.

What is claimed is:
 1. A method of treating a human patient diagnosedwith Lambert-Eaton myasthenic syndrome (LEMS) in need of treatmentthereof comprising administering a total daily dose of about 30 mg toabout 240 mg of 3,4-diaminopyridine (3,4-DAP), or an equivalent amountof a pharmaceutically acceptable salt thereof, to a human patientdiagnosed with LEMS having two N-acetyl transferase 2 (NAT2) fastalleles, wherein the total daily dose is optionally provided as a seriesof divided doses.
 2. The method of claim 1, wherein the two NAT2 fastalleles are wild-type alleles.
 3. The method of claim 1, wherein theabout 30 mg to about 240 mg of 3,4-DAP is provided via an equivalentamount of 3,4-DAP phosphate salt.
 4. The method of claim 1, wherein thetotal daily dose is about 30 mg to about 100 mg of 3,4-DAP, or anequivalent amount of a pharmaceutically acceptable salt thereof.
 5. Themethod of claim 4, wherein the total daily dose is about 30 mg to about80 mg of 3,4-DAP, or an equivalent amount of a pharmaceuticallyacceptable salt thereof.
 6. The method of claim 5, wherein the totaldaily dose is about 30 mg to about 50 mg of 3,4-DAP, or an equivalentamount of a pharmaceutically acceptable salt thereof.
 7. The method ofclaim 4, wherein the total daily dose is provided via up to 5 divideddoses per day.
 8. The method of claim 7, wherein the total daily dose isprovided via 3 to 4 divided doses per day.
 9. The method of claim 7,wherein the total daily dose is provided via 2 to 3 divided doses perday.
 10. The method of claim 4, wherein the total daily dose is providedas a series of divided doses, and each divided dose is about 10 mg,about 15 mg, about 20 mg, about 25 mg, or about 30 mg of 3,4-DAP, or theequivalent amount of a pharmaceutically acceptable salt thereof.
 11. Themethod of claim 4, wherein the total daily dose is provided as a seriesof divided doses, and each divided dose is provided as one or morescored tablets or portions thereof.
 12. The method of claim 11, whereinthe scored tablet comprises about 10 mg of 3,4-DAP, or the equivalentamount of a pharmaceutically acceptable salt thereof.
 13. The method ofclaim 4, wherein the total daily dose is provided as a series of divideddoses, and each divided dose is provided as an aqueous suspension. 14.The method of claim 4, wherein the total daily dose is about 30 mg of3,4-DAP, or the equivalent amount of a pharmaceutically acceptable saltthereof.
 15. The method of claim 4, wherein the total daily dose isabout 50 mg of 3,4-DAP, or the equivalent amount of a pharmaceuticallyacceptable salt thereof.
 16. The method of claim 4, wherein the totaldaily dose is about 60 mg of 3,4-DAP, or the equivalent amount of apharmaceutically acceptable salt thereof.
 17. The method of claim 4,wherein the total daily dose is about 80 mg of 3,4-DAP, or theequivalent amount of a pharmaceutically acceptable salt thereof.
 18. Themethod of claim 4, wherein the total daily dose is about 100 mg of3,4-DAP, or the equivalent amount of a pharmaceutically acceptable saltthereof.
 19. A method of treating a human patient diagnosed with LEMS inneed of treatment thereof comprising administering a total daily dose ofabout 30 mg to about 100 mg of 3,4-DAP, or an equivalent amount of apharmaceutically acceptable salt thereof, as a series of divided dosesto a human patient diagnosed with LEMS having two NAT2 fast alleles. 20.The method of claim 19, wherein the two NAT2 fast alleles are wild-typealleles.
 21. The method of claim 19, wherein the total daily dose isprovided via up to 5 divided doses per day.
 22. The method of claim 21,wherein the total daily dose is about 30 mg of 3,4-DAP, or theequivalent amount of a pharmaceutically acceptable salt thereof.
 23. Themethod of claim 21, wherein the total daily dose is about 50 mg of3,4-DAP, or the equivalent amount of a pharmaceutically acceptable saltthereof.
 24. The method of claim 21, wherein the total daily dose isabout 60 mg of 3,4-DAP, or the equivalent amount of a pharmaceuticallyacceptable salt thereof.
 25. The method of claim 21, wherein the totaldaily dose is about 80 mg of 3,4-DAP, or the equivalent amount of apharmaceutically acceptable salt thereof.
 26. The method of claim 21,wherein the total daily dose is about 100 mg of 3,4-DAP, or theequivalent amount of a pharmaceutically acceptable salt thereof.
 27. Amethod of treating a human patient diagnosed with LEMS in need oftreatment thereof comprising administering a total daily dose of 3,4-DAPphosphate salt, which is equivalent to a total daily dose of about 30 mgto about 100 mg of 3,4-DAP, as a series of divided doses to a humanpatient diagnosed with LEMS having two NAT2 fast alleles.
 28. The methodof claim 27, wherein the two NAT2 fast alleles are wild-type alleles.